Fluorescent universal nucleic acid end label

ABSTRACT

Structural analogs of the six non-fluorescent N-nucleosides commonly found in RNA and DNA, which are inherently fluorescent under physiological conditions, are identified and methods for their preparation provided. Such analogs may be incorporated into DNA and/or RNA oligonucleotides via either enzymatic or chemical synthesis to produce fluorescent oligonucleotides having prescribed sequences. Such analogous sequences may be identical to, or the analogous complement of, template or target DNA or RNA sequences to which the fluorescent oligonucleotides can be hybridized. Methods of preparing either RNA or DNA oligonucleotide probes of the invention, intermediates used in such methods, and methods of using the probes of the invention in oligonucleotide amplification, detection, identification, and/or hybridization assays are also provided.

CROSS REFERENCES TO RELATED APPLICATIONS

This application is a divisional of application Ser. No. 08/292,892,filed Aug. 18, 1994, which was a continuation of application Ser. No.08/108,457, filed Aug. 18, 1993, abandoned, which was a continuation ofapplication Ser. No. 08/021,539, filed Feb. 12, 1993, abandoned, whichwas a continuation of application Ser. No. 07/834,456, filed Feb. 12,1992, abandoned.

BACKGROUND OF THE INVENTION

A. Field of the Invention

The present invention relates to fluorescent structural analogs of thenon-fluorescent nucleosides commonly found in DNA and RNA, methods oftheir derivatization and subsequent use in the synthesis of fluorescentoligonueleotides, and to their new and useful applications both asfluorescent monomers and in fluorescent oligonucleotides havingprescribed sequences. Additionally, it relates to applications in whichfluorescent structural analogs are substituted for specificnon-fluorescent nucleosides in prescribed DNA or RNA sequences and tomethods of using fluorescent oligonucleotides as hybridization reagentsand probes for diagnostic and therapeutic purposes and as diagnostic andtherapeutic research tools.

B. General Description of the Art

The six commonly occurring N-nucleosides which predominate in thecomposition of DNA and RNA from all sources have the structures shown inFIG. 1 wherein R₆ is H for inosine and NH₂ for guanosine, R₉ is H foruridine and CH₃ for thymidine. Furthermore, R₁₂, R₁₄ ═OH forribonucleotides, R₁₂ ═OH, R₁₄ ═H for 2'-deoxy nucleotides, R₁₂ ═H, R₁₄═OH for 3'-deoxy nucleotides, and R₁₂, R₁₄ ═H in dideoxy nucleotides.

The six commonly occurring nucleotides do not absorb light atwavelengths >290 nm and are effectively non-fluorescent underphysiological conditions. Derivatives of the commonly occurringN-nucleotides for a variety of synthetic, diagnostic, and therapeuticpurposes are common, including substitutions on both the heterocyclicbase and the furanose ring. These substitutions can be made at the locishown in FIG. 2 in which R₄ is a reactive group derivatizible with adetectable label (NH₂, SH, ═O, and which can include an optional linkingmoiety including, but not limited to, an amide, thioether, or disulfidelinkage or a combination thereof with additional variable reactivegroups, R₁ through R₃, e.g., R₁ --(CH₂)_(x) --R₂, or R₁ -R₂ -(CH₂)_(x)--R₃ --, where x is an integer in the range of 1 and 25 inclusive; andR₁, R₂, and R₃ can be H, OH, alkyl, acyl, amide, thioether, ordisulfide); R₅ is H or part of an etheno linkage with R₄ ; R₆ is H, NH₂,SH, or ═O; R₉ is hydrogen, methyl, bromine, fluorine, or iodine, or analkyl or aromatic substituent, or an optional linking moiety includingan amide, thioether, or disulfide linkage or a combination thereof suchas R₁ --(CH₂)_(x) --R₂, or R₁ --R₂ --(CH₂)_(x) --R₃ --, where x is aninteger in the range of 1 and 25 inclusive; R₁₀ is hydrogen, or anacid-sensitive base stable blocking group, or a phosphorous derivative,R₁₁ ═R₁₂ ═H; R₁₂ is hydrogen, OH, or a phosphorous derivative; R₁₄ is H,OH, or OR₃ where R₃ is a protecting group or additional fluorophore. Theletters N and C in the N-nucleosides and C-nucleosides designate theatom at which the glycosidic covalent bond connects the sugar and theheterocyclic base. In the cases of the commonly occurring nucleosides,the bases are either adenine, guanine, cytosine, inosine, uracil, orthymine. The bases are attached to a furanose sugar, a general structureof which is shown in FIG. 3. The sugar substituents for the fluorescentanalogs share the same numbering system for all R groups, but thenumbering system for some of the heterocycle analogs may differ.

I. Known Methods of Labeling Nucleotides

Nucleotide sequences are commonly utilized in a variety of applicationsincluding diagnostic and therapeutic probes which hybridize target DNAand RNA and amplification of target sequences. It is often necessary, oruseful, to label nucleotide sequences.

A. Labeling of oligonucleotide probes with radioisotopes. Hybridizationof specific DNA or RNA sequences typically involves annealingoligonucleotides of lengths which range from as little as 5 bases tomore than 10,000 bases (10 kb). The majority of oligonucleotide probescurrently in research use are radioactively labeled; however, because of(a) the short half lives of the isotopes in common usage, (b) the safetyrequirements, and (c) the costs of handling and disposal of radioactiveprobes, convenient and sensitive non-isotopic methods of detection arerequired for hybridization diagnostic methods to achieve widespreadacceptance and application.

B. Non-isotopic methods of labeling oligonucleotide probes. In general,all of the non-isotopic methods of detecting hybridization probes thatare currently available depend on some type of derivatization of thenucleotides to allow for detection, whether through antibody binding, orenzymatic processing, or through the fluorescence or chemiluminescenceof an attached "reporter" molecule. In most cases, oligonucleotides havebeen derivatized to incorporate single or multiple molecules of the samereporter group, generally at specific cyclic or exocyclic positions.Techniques for attaching reporter groups have largely relied upon (a)functionalization of 5' or 3' termini of either the monomericnucleosides or the oligonucleotide strands by numerous chemicalreactions using deprotected oligonucleotides in aqueous or largelyaqueous media (see Cardullo et al. 1988! PNAS 85: 8790-8794); (b)synthesizing modified nucleosides containing (i) protected reactivegroups, such as NH₂, SH, CHO, or COOH, (ii) activatable monofunctionallinkers, such as NHS esters, aldehydes, or hydrazides, or (iii) affinitybinding groups, such as biotin, attached to either the heterocyclic baseor the furanose moiety. Modifications have been made on intactoligonucleotides or to monomeric nucleosides which have subsequentlybeen incorporated into oligonucleotides during chemical synthesis viaterminal transferase or "nick translation" (see, e.g., Brumbaugh et al.1988!PNAS 85: 5610-5614; Sproat, B. S., A. I. Lamond, B. Beijer, P.Neuner, P. Ryder 1989!Nucl. Acids Res. 17: 3371-3385; Allen, D. J., P. LDarke, S. J. Benkovic 1989!Biochemistry 28: 4601-4607); (c) use ofsuitably protected chemical moieties, which can be coupled at the 5'terminus of protected olignnucleotides during chemical synthesis, e.g.,5'-aminohexyl-3'-O-phosphoramidite (Haralambidis, J., L. Duncan, G. W.Tregar 1990!Nucl. Acids Res. 18: 493-499); and, (d) addition offunctional groups on the sugar moiety or in the phosphodiester backboneof the polymer (see Conway, N. E., J. Fidanza, L. W. McLaughlin1989!Nucl. Acids Res. Symposium Series 21: 43-44; Agrawal, S., P. C.Zamecnik 1990! Nucl. Acids Res. 18: 5419-5423).

At the simplest, non-nucleoside linkers and labels have been attached tothe 3' or 5' end of existing oligonucleotides by either enzymatic orchemical methods. Modification of nucleoside residues internal to thesequence of a DNA or RNA strand has proven to be a difficult procedure,since the reaction conditions must be mild enough to leave the RNA orDNA oligomers intact and still yield reaction products which canparticipate in normal Watson-Crick base pairing and stackinginteractions (see FIG. 4).

C. Derivatizations of the heterocyclic base (B). Numerous methods forboth cyclic and exocyclic derivatization of the N-nucleoside base havebeen described, including the following:

(1) Hapten labeling. DNA probes have been amino modified andsubsequently derivatized to carry a hapten such as 2,4-dinitrophenol(DNP) to which enzyme-conjugated anti-hapten antibodies bind whichsubsequently can be processed using a colorimetric substrate as a label(Keller et al. 1988!Analytical Biochemistry 170: 441-450).

(2) Amino- and thiol-derivatized oligonueleotides. Takeda and Ikeda (1984! Nucl. Acids Research Symposium Series 15: 101-104) usedphosphotriester derivatives of putresceinyl thyroidine for thepreparation of amino-derived oligomers. Ruth and colleagues havedescribed methods for synthesizing a deoxyuridine analog with a primaryamine "linker arm" 12 carbons in length at C₅ (Jablonski et al. 1986!Nucl. Acids Res. 14: 6115-6128). These were later reacted withfluorescein to produce a fluorescent molecule. Urdea and Horn weregranted a patent in 1990 (U.S. Pat. No. 4,910,300) covering pyrimidinederivatives on which the 6-amino group at C₄ had been modified. 3' and5' amino modifying phosphoramidites have been widely used in chemicalsynthesis or derivatized oligonucleotides and are commerciallyavailable.

(3) Labeling with photobiotin and other biotinylating agents. The highaffinity of biotin for avidin has been used to bind enzymatic orchemiluminescent reagents to derivatized DNA probes (Foster et al.1985!Nucl. Acids Res. 13: 745-761). Biotin conjugated to other linkershas also been widely used, including biotin-NHS esters (Bayer, E. A., M.Wilchek 1980! Methods in Biochemical Analysis 26: 1), biotinsuccinamides (Lee, W. T., D. H. Conrad 1984! J. Exp. Med. 159: 1790),and biotin maleimides (Bayer, E. A. et al. 1985! Anal. Biochem. 149:529). Reisfeld et al. ( 1987! BBRC 142: 519-526) used biotin hydrazideto label the 4-amino group of cytidine. A patent was granted to Klevanet al. in 1989 (U.S. Pat. No. 4,828,979) for such derivatizations at the6-position of adenine, the 4-position of cytosine, and the 2-position ofguanine. These derivatizations interfere with hydrogen bonding andbase-pairing and have limited uses in producing oligomers for use inhybridization.

(4) dU-Biotin labeling. Nucleoside 5'-triphosphates or3'-O-phosphoramidites were modified with a biotin moiety conjugated toan aliphatic amino group at the 5-position of uracil (Langer et al.1981!PNAS 78: 6633-6637; Saiki et al. 1985! Science 230: 1350-1354). Thenucleotide triphosphate derivatives are effectively incorporated intodouble stranded DNA by standard techniques of "nick translation." Oncein an oligonucleotide, the residue may be bound by avidin, streptavidin,or anti-biotin antibody which can then be used for detection byfluorescence, chemiluminescence, or enzymatic processing.

(5) 11-digoxigenin-ddUTP labeling. The enzyme, terminal transferase, hasbeen used to add a single digoxigenin-11-dideoxyUTP to the 3' end ofoligonucleotides. Following hybridization to target nucleic acids,DIG-ddUTP labeled hybridization probes were detected using anti-DIGantibody conjugate.

(6) AAIF. Immunofluorescent detection can be done using monoclonal Fab'fragments which are specific for RNA:DNA hybrids in which the probe hasbeen derivatized with, e.g., biotin-11-UTP (Bobo et al. 1990! J. Clin.Microbial. 28: 1968-1973; Viscidi et al. 1986!J. Clin. Microbiol. 23:311-317).

(7) Bisulfite modification of cytosine. Draper and Gold ( 1980!Biochemistry 19: 1774-1781) introduced aliphatic amino groups ontocytidine by a bisulfite catalyzed termination reaction; the amino groupswere subsequently labeled with a fluorescent tag. In this procedure, theamino group is attached directly to the pyrimidine base. Like thederivatization of uracil, these derivatizations interfere with hydrogenbonding and base-pairing and are not necessarily useful for producingefficient hybridization oligomers.

(8) Fluorophore derivatized DNA probes. Texas Red(Sulfochloro-Rhodamine) derivatized probes are commercially availablewhich hybridize to specific target DNAs and which can be detected usinga flow cytometer or a microscope. Numerous authors have reportedcoupling fluorophores to chemically synthesized oligonucleotides whichcarried a 5' or 3' terminal amino or thiol group (Brumbaugh et al. 1988!Nucleic Acids Res. 16: 4937-4956).

(9) Direct enzyme labeling. Chemical coupling of an enzyme directly to achemically synthesized probe has been used for direct detection throughsubstrate processing. For example, Urdea et al. described anoligonucleotide sandwich assay in which multiple DNA probehybridizations were used to bind target DNA to a solid phase after whichit was further labeled with additional, alkaline phosphatase-derivatizedhybridization probes (Urdea et al. 1989! Clin. Chem. 35: 1571-1575).

(10) Acridinium ester labeling. A single phenyl ester of methylacridinium is attached at a central position on an RNA or DNA probe.Hydrolysis of the ester releases an acridone, CO₂, and light. Becausethe ester on unhybridized probes hydrolyzes more quickly than the esteron probes which have hybridized to target RNA or DNA, thechemiluminescence of the hybridized probes can be distinguished fromthat of free probes and is used in a "hybridization protection assay"(Weeks et al. 1983!Clin. Chem. 29: 1474-1479).

D. Derivatizations of the furanose ring (F). Methods for derivatizationof the furanose ring (R₁₁ through R₁₄ in FIG. 3) and at thephosphodiester backbone of oligonucleotides (R₁₀ in FIG. 3) have beenreported.

(1) Internucleotide linkage reporter groups (R₁₀ site). Phosphorothioateesters have been used to provide a binding site for fluorophores such asmonobromobimane (Conway et al. 1989!Nucl. Acids Res. Symposium Series21: 43-44). Agrawal and Zamecnik ( 1990! Nucl. Acids Res. 18: 5419-5423)reported methods for incorporating amine specific reporter groups (e.g.,monobromobimane) and thiol specific reporter groups (e.g., fluoresceinisothiocyanate) through modifying the phosphodiester backbone of DNA tophosphoramidites and phosphorothioate diesters, respectively.

(2) Glycosidic reporter groups (R₁₁ through R₁₄ sites). Smith, Fung, andKaiser ( 1989! U.S. Pat. No. 4,849,513) described syntheses for anassortment of derivatives and labels on the glycosidic moiety ofnucleosides and nucleoside analogs through the introduction of analiphatic amino group at R₁₀. The authors did not report or claim anyuses or applications of inherently fluorescent oligonucleotides, eithermade chemically or enzymatically or using the fluorescent nucleosideanalogs or their derivatives.

E. Limitations of non-isotopic methods for labeling oligonucleotides. Inorder to create non-radioactive types of detectable oligonucleotides, ithas been necessary to chemically modify the nucleosides typically usedin DNA and RNA probes, which has made such probe preparation expensiveand laborious; in many cases the detection chemistries have also provencumbersome and expensive to use, which has largely been responsible fortheir failure to find significant application in clinical laboratories.In their applications to hybridization, other limitations of chemicallyderivatized probes have also become apparent.

(1) Chemically derivatized dNTPs are generally not cost-effective foruse as stock deoxynucleotide triphosphates in PCR amplification, hence,labeling of amplified DNA is limited to (i) amplification usingpreviously labeled primers, or (ii) annealing with labeled hybridizationprobes. Use of the former frequently results in false positives duringamplification owing to (i) non-specific annealing of primers tonon-target segments of DNA during amplification, or (ii) contaminationby amplicons present in the laboratory environment which are residualfrom previous amplification experiments. Expense and technicaldifficulties in post-hybridization processing have largely limited theapplications of labeled hybridization probes to research.

(2) Base pairing is hindered for many oligomers made with derivatizednucleosides through the introduction of bulky or non-hydrogen bondingbases at inappropriate sites in a sequence. Owing to the inherentbackground chemiluminescence of many clinical samples, even theacridinium ester probes have failed to achieve their theoretical levelsof sensitivity. The requirements for post hybridization processing haveremained a limitation to such methods.

(3) It has proven difficult to provide non-radioactively labeled probeswhich may be inexpensively produced in large quantities.

(4) Chemiluminescent probes are short lived and samples so tested aredifficult to quantify or to "reprobe" accurately.

(5) Hybridization in most cases is only inferred, is non-quantitative oronly semi-quantitative, and is non-automatable.

These limitations have hindered applications of DNA and RNAhybridization probes to clinical laboratory testing and therapeuticuses.

F. Fluorescent N-nucleosides and fluorescent structural analogs.Formycin A (generally referred to as Formycin), the prototypicalfluorescent nucleoside analog, was originally isolated as an antitumorantibiotic from the culture filtrates of Nocardia interforma (Hori etal. 1966! J. Antibiotics, Set. A 17: 96-99) and its structure identifiedas 7-amino-3-b-D-ribafuranosyl (1H-pyrazolo- 4,3d!pyrimidine)) (FIGS. 5and 6). This antibiotic, which has also been isolated from culturebroths of Streptomyces lavendulae (Aizawa et al. 1965! Agr. Biol. Chem.29: 375-376), and Streptomyces gummaensis (Japanese Patent No. 10,928,issued in 1967 to Nippon Kayaku Co., Ltd.), is one of numerous microbialC-ribonucleoside analogs of the N-nucleosides commonly found in RNA fromall sources. The other naturally-occurring C-ribonucleosides which havebeen isolated from microorganisms (FIG. 4) include formycin B (Koyama etal. 1966! Tetrahedron Lett. 597-602; Aizawa et al., supra; Umezawa etal. 1965! Antibiotics Ser. A 18: 178-181), oxoformycin B (Ishizuka etal. 1968! J. Antibiotics 21: 1-4; Sawa et al. 1968!Antibiotics 21:334-339), pseudouridine (Uematsu and Suahdolnik 1972! Biochemistry 11:4669-4674), showdomycin (Darnall et al. 1967! PNAS 57: 548-553),pyrazomycin (Sweeny et al. 1973!Cancer Res. 33: 2619-2623), andminimycin (Kusakabe et al. 1972! J. Antibiotics 25: 44-47). Formycin,formycin B, and oxoformycin B are pyrazolopyrimidine nucleosides and arestructural analogs of adenosine, inosine, and hypoxanthine,respectively; a pyrazopyrimidine structural analog of guanosine obtainedfrom natural sources has not been reported in the literature. A thoroughreview of the biosynthesis of these compounds is available in Ochi etal. (1974) J. Antibiotics xxiv: 909-916.

Physical properties of the nucleoside analogs. Because several of theC-nucleosides were known to be active as antibiotic, antiviral, oranti-tumor compounds, their chemical derivatization and physicalproperties have been extensively studied and compared to the structuresand syntheses of the N-nucleosides commonly found in DNA and RNA. In thelate 1960s, several structural analogs of the six commonly occurringN-nucleosides were found to be fluorescent under physiologicalconditions; fluorescence in the analogs results from a molecularrigidity of the heterocycle structure itself; not all the structuralanalogs of a given type, e.g., the C-nucleosides, are fluorescent, noris fluorescence an exclusive or inherent property of any particularclass of structural analogs. Our subsequent studies have shown that onlya few of the pyrazolo and pyrolo pyrimidines and purines arefluorescent, and that the property is shared with a few other nucleosidederivatives and structural analogs including, but not limited to,several substituted N-nucleosides, azanucleosides, ethenonucleosides,and deazanucleosides, the structures of which are shown in FIGS. 5-11.Those structures in FIGS. 5-11 which are shown surrounded by boxes havebeen either previously reported or found to be fluorescent duringdevelopment of the present invention.

Uncharacterized oligomers containing fluorescent analogs were preparedby Ward and colleagues for physical studies using then availablenucleoside polymerase enzymes (Ward et al. 1969! J. Biol. Chem. 244:3243-3250; Ward et al. 1969!loc cit 1228-1237). There have been norecent reports in the literature of attempts to combine the use offluorescent nucleosides or their structural analogs with the synthesisor hybridization techniques of molecular biology or to synthesizefluorescent oligonucleotides therefrom.

BRIEF SUMMARY OF THE INVENTION

The subject invention pertains to nucleoside analogs which arefluorescent. These fluorescent nucleoside analogs are useful as monomersin synthesizing and labelling nucleotide sequences. The inventionfurther pertains to the use of these fluorescent nucleotides which canbe substituted for naturally occurring nucleosides in the synthesis ofoligonucleotide probes. When used as hybridization probes, thefluorescence of such oligonucleotides can be used as a diagnostic toolto detect and identify specific genetic sequences. This methodology isdistinct from other non-radioactive methods of probe detection in thatit does not utilize nucleotides which have been coupled to enzymes orother reactive proteins and does not require post-hybridizationprocessing for the detection of hybridization.

As described in the Background section, there are many shortcomings tothe methods and compositions currently used in DNA and RNA probetechnology. It is an object of the present invention to overcome theseshortcomings of the prior art through the use of fluorescent nucleosidesand their fluorescent structural analogs which can be directlyincorporated into a prescribed sequence as (i) specific substitutes fora given nonfluorescent nucleotide which appear at defined locations inthe complementary sequences to template or target DNA, and (ii) aslabels for the identification and detection of specific sequences oftemplate, product, amplified, or target DNA and/or RNA.

It is another object Of the present invention to provide novel,inherently fluorescent nucleoside and nucleoside analogs and the noveltriphosphate and phosphoramidite forms thereof, which are useful in thesynthesis of labeled polynucleotide probes, amplimers, diagnostics, andtherapeutics. It is a further object of the present invention to providemethods of making autofluorescent oligonucleotides capable of specificWatson-Crick base pairing with prescribed sequences of target DNA orRNA.

It is another object of the invention to provide methods of usingfluorescent nucleoside analogs and oligonucleotides made therefrom andsynthesized according to the methods of the present invention toidentify, detect the presence of, and/or alter the function of knownnucleic acid sequences of DNA and RNA. Additionally, it is an object toimprove and simplify the methods of detection, and to simplify theapplications and uses of DNA and RNA hybridization techniques.

In another aspect of the invention, enzymatic methods are provided formaking nucleic acid probes which are complementary to, and will bind to,only the sense or only the anti-sense, but not both, strands of a DNAduplex (asymmetric synthesis). It is an important aspect of theinvention that asymmetric synthesis is the necessary condition forcreating rapid and quantitative nucleic acid probe tests, assays,diagnostics, and therapeutics. A significant aspect of asymmetricsynthesis is its dependence on the asymmetric use of promoters, primers,or linker modified primers to direct the synthesis or isolation ofoligonueleotides or oligomers using only one of the two strands of aduplex as the template.

It is yet another aspect of the invention that asymmetric synthesismakes possible the directed use of multiple different templates forconcurrent synthesis of a "cocktail" of asymmetric probes which canhybridize concurrently to independent and unique target sites on asingle piece of nucleic acid, genomic DNA, or chromosome. It is animportant aspect of the invention of probe "cocktails" that if multiplecopies of the same target sequence are present on a single genome, suchas the multiple copies of the tandem repeat intergenic sequencesdisclosed in Example 7, a single asymmetric probe template can be usedto create a "cocktail" which will bind to many targets on a singlegenome which are identical in sequence but widely distributed in locuson the genome.

In one aspect of the invention, fluorescent structural analogs of thecommonly occurring nucleosides and their derivatives useful in thesynthesis, labeling, and detection of oligonucleotides are providedhaving the structural formulae of FIGS. 5 through 11. The commonlyoccurring nucleosides characteristically form hydrogen bonds in aspecific donor/acceptor relationship, designated Watson-Crick basepairing as shown in FIG. 4. Where appropriate, specific fluorescentnucleoside analogs capable of reproducing the pattern of Watson-Crickhydrogen bond formation analogous to that of a particular commonlyoccurring nucleoside are provided, as indicated for, e.g., A:T andformycin:T in FIG. 4 by the donor/acceptor patterns.

In another aspect of the invention, methods of making and derivatizingthe fluorescent structural analogs of the commonly occurring nucleosidesare provided including the steps of derivatizing the R₁₀, R₁₂, and R₁₄moieties to be (i) reactive in DNA or RNA synthesis, and/or (ii)reactive in Resonance Energy Transfer of the fluorescence from thestructural analogs.

In still another aspect, methods of synthesizing and usingpolynucleotide probes are provided using one or more of the fluorescentstructural analogs and/or their derivatized forms. Such probes can beused to screen a sample containing a plurality of single stranded ordouble stranded polynucleotide chains and will label, detect, andidentify the desired sequence, if present, by hybridization. It is animportant aspect of the invention that the fluorescent oligonucleotideprobes can be used with "solution hybridization" methods as depicted inFIGS. 12 through 18.

In accordance with the foregoing objects, the present inventioncomprises inherently fluorescent nucleosides which can be used to label,modify, or identify oligonucleotides made therefrom, the uses of suchinherently fluorescent oligonucleotides as hybridization probes, andmethods for detecting nucleotide sequences.

An important aspect of the invention is the stable fluorescence emissionof the fluorophores and the use of time-resolved spectroscopy or photoncounting to detect and to quantify the amount of a fluorophore presentin a sample.

Additional formulae, advantages, methods of use, and novel features ofthe invention will be set forth in the description which follows, and inpart become apparent to those skilled in the art after examination ofthe following, or may be learned by practice of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the six commonly-occurring N-nucleosides which predominatein DNA and RNA.

FIG. 2 shows the general structures of the commonly-occurringN-nucleosides and their derivatization sites, R_(n).

FIG. 3 shows the general structure of the faranose ring of both thepurine and pyrimidine nucleosides and the common sites, R_(n) forderivatization.

FIG. 4 shows Watson-Crick base pairing between the normally occurringN-nucleotides A:T and G:C and base pairing between formycin:T,formyein:U, 2,6-diaminopurine:T, and 5-amino-formycin B:C.

FIG. 5 shows structural analogs of the commonly-occurring N-nucleosidesderived from biological sources.

FIG. 6 shows the pyrazolo 4,3d!pyrimidine nucleoside analogs.

FIG. 7 shows the pyrazolo 3,4d!pyrimidine nucleoside analogs.

FIG. 8 shows the pyrazolo 1,5a!-1,3,5-triazine nucleoside analogs.

FIG. 9 shows the azapyrimidine and azapurine nucleoside analogs.

FIG. 10 shows the deazapyrimidine and deazapurine nucleoside analogs.

FIGS. 11A-11B shows examples of some fluorescent structural analogswhich are (11A) non-H-binding, and (11B) fluorescence resonance energytransfer (FRET) analogs.

FIG. 12 is a diagram of symmetric RNA synthesis using FTP or ATP.

FIG. 13 is a diagram of promoter directed asymmetric RNA probe synthesisusing viral promoters and vital RNA polymerases.

FIG. 14 is a diagram showing an example of the method for one-steplabeling of ssDNA inserted at the EcoRI site of pUG/M13 plasmid vectorsand using dF₁₀₅.

FIG. 15 is a diagram showing the necessity of using asymmetric DNA orRNA probes for rapid and quantitative hybridization of the probe totarget DNA. As shown, asymmetric probes provide significant increases inhybridization efficiencies when compared with symmetric probes.

FIG. 16 is a diagram showing the conversion of the ribonucleosideanalog, formycin A, to its 2'-deoxy triphosphate or phosphoramiditeforms.

FIG. 17 is a diagram of detection of a target DNA sequence in genomicDNA hybridization with fluorescent probes.

FIG. 18 is a diagram of detection of an amplified DNA segment bysolution hybridization of a fluorescent probe.

FIG. 19 shows a flow chart diagramming the separation scheme used toseparate reaction products from unreacted reagents following theenzymatic substitution reaction of FTP for ATP in RNA probes.

FIG. 20 shows a schematic of the mechanism for increasing detectionsensitivity by the use of a probe "cocktail" which contains multipleprobes of different sequences.

FIGS. 21A-21L show specific fluorescent nucleoside analogs which havebeen identified and characterized as to their class, structure, chemicalname, absorbance spectra, emission spectra, and methods of synthesis.

FIG. 22 shows the 5' universal end label comprising four distinctfunctional groups which include: region A, a non-base-pairinghomopolymer from 1 to about 50 fluorescent nucleoside analogs long;region B, an optional non-nucleoside phosphodiester "tether" connectingregion A to region C; region C, an enzymatic synthesis primer,complementary to a promoter in the target sequence; and region D, anucleotide chain from about 40 to about 20,000 nucleotides in lengthcomplementary to the target nucleotide sequence. Regions A, B, and Cwhich are typically 20 to 60 bases in length can be chemicallysynthesized. Region D can preferably be enzymatically synthesized. Thesynthesis of the 5' universal end label is described in Example 8.

FIG. 23 shows a schematic representation of the method for synthesizinga highly flourescent 5' labeled probe. The method comprises thefollowing steps: (1) restricting, with a specific restriction enzyme, asequence having a known promoter site and known restriction sitedownstream from the known promoter site, (2) inserting a unique targetsequence at the restriction site, (3) hybridizing a fluorescentnucleoside analog probe comprising a sequence complementary to thepromoter of the inserted target sequence, and (4) extending the probesequence from the hybridized promoter region, using a nucleic acidpolymerase, to synthesize a specific probe complementary to the insertedtarget sequence.

FIG. 24 shows the normalized spectrum profiles compared for F₁₈₅(ethenoadenosine) and F₁₀₅ (formycin). The spectra were determinedbetween 250-405 nm for the compounds, using a 2.5 nm slit.

FIG. 25 shows a method for increasing sensitivity of detection ordifferential labeling using multiple copies of a 5' universal end label.Shown is an example of a target sequence, using the 1361 base pairIS6110 repeat from Mycobacterium tuberculosis. The non-hybridizedportion of the probe is the 5' universal end label on each of aplurality of probe sequences where each of the probe sequences iscomplementary to a different fragment or segment of the target sequence.

FIG. 26 shows the sequencing application of 5' universal end label. Theunique probe (which can be produced by the method shown in FIG. 24) canbe employed using DNA polymerase to produce a plurality of dideoxyfragments having different lengths.

FIG. 27 shows the Sustained Signal Amplification procedure. The examplein the figure employs the genome from the Hepatitis B virion having twounequal lengths of DNA forming its double-stranded genome. The methodshows the steps: (1) extension of the shorter DNA strand usingnucleotides or phosphorylated nucleoside analogs; (2) the separation ofthe two strands and the addition of two primers, A and B; (3) extensionof the DNA strand to which the A primer is hybridized using reversetranscriptase; (4) utilization of the synthesized double-strandedsequence in an amplification cycle which comprises (a) production ofantisense RNA template from the synthesized DNA using an RNA polymerase,(b) synthesis of DNA from the RNA template using primer B and a nucleicacid polymerase, and (c) synthesis of double stranded DNA (DNAreplicate) from the B primer-DNA. The replicate DNA can then repeat thesignal amplification cycle (step (4)).

BRIEF DESCRIPTION OF THE SEQUENCES

SEQ ID NO. 1 is a synthetic oligonucleotide according to the subjectinvention.

SEQ ID NO. 2 is a synthetic oligonucleotide and the complement of SEQ IDNO. 1.

SEQ ID NO. 3 is a synthetic oligonucleotide and a fluorescent analog ofSEQ ID NO. 2.

DETAILED DISCLOSURE OF THE INVENTION

Disclosed and claimed are novel fluorescent nucleoside analogs andmethods of use of the fluorescent nucleosides in, for example, nucleicacid probes and diagnostic kits. One preferred embodiment pertains tothe use of inherently fluorescent nucleoside analogs in the chemical andenzymatic synthesis of DNA hybridization probes including solid phasesynthesis, template directed enzymatic polymerization and amplificationusing polymerase chain reaction methods. Another embodiment relates tothe use of autofluorescent DNA hybridization probes in theidentification of specific DNA sequences, e.g., gene mapping and thedetection and diagnosis of infectious and genetic diseases.

Specifically, the subject invention pertains to nucleoside analogs whichare fluorescent and which can be substituted for naturally occurringnucleosides in the synthesis of oligonucleotide probes. When used ashybridization probes, the fluorescence of such oligonucleotides can beused in a variety of procedures to detect and identify specific geneticsequences. This methodology is distinct from other non-radioactivemethods of probe detection in that it does not utilize nucleotides whichhave been coupled to enzymes or other reactive proteins. Thus, describedherein are applications of inherently fluorescent nucleoside analogs indeveloping hybridization techniques for routine, automatable clinicaldiagnosis.

The fluorescent analogs of the subject invention are of three generaltypes: (A) C-nucleoside analogs; (B) N-nucleoside analogs; and (C)N-azanucleotide and N-deazanucleotide analogs. All of these compoundshave three features in common: 1) they are structural analogs of thecommon nucleosides capable of replacing naturally occurring nucleosidesin enzymatic or chemical synthesis of oligonucleotides; 2) they arenaturally fluorescent when excited by light of the appropriatewavelength(s) and do not require additional chemical or enzymaticprocesses for their detection; and 3) they are spectrally distinct fromthe nucleosides commonly encountered in naturally occurring DNA. Atleast 125 specific compounds of the subject invention have beenidentified. These compounds, which have been characterized according totheir class, structure, chemical name, absorbance spectra, emissionspectra, and method of synthesis, are tabulated as shown in FIGS.21A-21F-1.

Definitions. The following definitions are provided for ease inunderstanding the description:

"Commonly Occurring Nucleosides" are the six monomeric N-nucleotidesshown in FIG. 1, which predominate in naturally occurring DNA and RNA,enter into classical Watson-Crick base pairing, and are effectivelynon-fluorescent under physiological conditions. The respectiveone-letter symbols in sequence shorthand are A, C, G, T, U, and I foradenosine, cytidine, guanidine, thymidine, uridine, and inosine,respectively.

"Structural Analogs" of the commonly occurring nucleosides arestructurally related molecules that mimic the normal purine orpyrimidine bases in that their structures (the kinds of atoms and theirarrangement) are similar to the commonly occurring bases, but may havecertain modifications or substitutions which do not affect basicbiological activity or biochemical functions. Such base analogs include,but are not limited to, imidazole and its 2,4- and/or 5-substitutedderivatives; indole and its 2-, 3-, 4-, 5-, 6-, and/or 7-substitutedderivatives; benzimidazole and its 3-, 4-, and/or 5-substitutedderivatives; indazole and its 3-, 4-, 5-, 6-, and/or 7-substitutedderivatives; pyrazole and its 3-, 4-, and/or 5-substituted derivatives;triazole and its 4- and/or 5-substituted derivatives; tetrazole and its5-substituted derivatives; benzotriazole and its 4-, 5-, 6-, and/or7-substituted derivatives; 8-azaadenine and its substituted derivatives;6-azathymine and its substituted derivatives; 6-azauracil and itssubstituted derivatives; 5-azacytosine and its substituted derivatives;8-azahypoxanthine and its substituted derivatives; pyrazolopyrimidineand its substituted derivatives; 3-deazauracil; orotic acid;2,6-dioxo-1,2,3,6-tetrahydro-4-pyrimidine carboxylic acid; barbituricacid; uric acid; ethenoadenosine; ethenocytidine; an allopurinol(4-hydroxy-pyrazolo 3,4d!pyrimidine); or their protected derivatives asdescribed below. Base analogs can also be any of the C-nucleosides suchas are shown in FIGS. 4 and 5 in which the normal C-N bond between thebase and the furanose ring is replaced by a C-C bond; such basesinclude, but are not limited to, uracil, as in the C-nucleosidepseudouridine; 1-methyluracil; 1,3-dimethyluracil;5(4)-carbomethoxy-1,2,3-triazole; 5(4)-carboxamido-1,2,3-triazole;3(5)-carboxymethylpyrazole; 3(5)-carbomethoxypyrazole;5-carboethoxy-1-methylpyrazole; maleimide (in the C-nucleosideshowdomycin); and 3(4)-carboxamido-4(3)-hydroxypyrazole (in theC-nucleoside pyrazomycin); and any of the other analogs listed orinferred in FIGS. 5 through 11; or their protected derivatives.

"Fluorophore" refers to a substance or portion thereof which is capableof emitting fluorescence in a detectable range. For the fluorescentstructural analogs of the nucleotides, this fluorescence typicallyoccurs at wavelengths in the near ultraviolet (>300 nm) through thevisible wavelengths. Preferably, fluorescence will occur at wavelengthsbetween 300 nm and 700 nm and most preferably in the visible wavelengthsbetween 300 nm and 500 nm.

"Fluorescent Structural Analogs" are synthetic or biochemically derivedmonomeric structural analogs of the six commonly occurring N-nucleosides(FIG. 1), such as are depicted in FIGS. 5 through 11, which may or maynot be capable of classical Watson-Crick base pairing depending upon themonomeric structure and/or oligonucleotide in which they are used, butwhich are spectrally unique and distinct from the commonly occurringnucleosides in their capacities for selective excitation and emissionunder physiological conditions. For example, the C-nucleoside formycin Ais a structural analog of adenosine that can form equivalentdonor/acceptor hydrogen bonds, but which has an excitation maximum inoligonucleotides at 303 nm and an emission maximum at 405 nm (StokesShift=102 nm).

"Derivatized" nucleoside analogs are fluorescent structural analogs inwhich reactive or protective functional groups are bound, covalently orotherwise, at the R₄ through R₉ positions of the heterocycle and/or theR₁₀ (5'), the R₁₂ (3'), and R₁₄ (2') positions of the glycosidic moiety.Derivatives at the 2' glycosidic position may include fluorescenceresonance energy transfer (FRET) acceptors or donors which enhance oraccept and re-emit at longer wavelengths the inherent fluorescenceemission of the fluorescent structural analog itself.

A "polynucleotide," "oligonucleotide," or "oligomer" is a nucleotidechain structure containing at least two commonly occurring nucleotidesor fluorescent structural analogs. The "fluorescent oligonucleotideprobe" or "fluorescent hybridization probe" provided herein is anucleotide chain structure, as above, containing at least two monomers,at least one of which is fluorescent.

"Hybridization" is the painwise annealing through Watson-Crick basepairing of two complementary, single-stranded molecules (see FIG. 4),which may be DNA:DNA, DNA:RNA, or RNA:RNA, and in which the two strandsmay come from different sources. The annealing is specific (i) forcomplementary base pairs in which the hydrogen bond donors and acceptorsare oriented as in FIG. 4, and (ii) for the complementary geneticsequence of the specific gene, target DNA, or target RNA (hereinafter"target DNA/RNA") to which the probe is to be hybridized. Compare, forexample, the hydrogen bond pattern of adenosine and formycin (FIG. 4).

"DNA/RNA Melting Temperature" and "Tm" refer to the temperature at whichthe hydrogen bonds between hybridized strands of DNA or RNA aredisrupted and the strands disassociate into single strands, therebydisrupting the structure of the duplex or hybrid.

"Analogous fluorescent sequence" refers to the nucleoside sequence of apolynucleotide which has been synthesized by any of the enzymatic orchemical methods described in the present invention, but in whichfluorescent nucleoside analogs have been explicitly substituted forparticular commonly occurring nucleosides, e.g., the substitution offormycin A-5'-triphosphate (FTP) for adenosine-5'-triphosphate (ATP),when using RNA polymerase to produce RNA probes complementary to aprescribed DNA template. In an analogous fluorescent sequence, thefluorescent nucleoside analog has been substituted in theoligonucleotide chain at some or all positions in which thecorresponding commonly occurring nucleotide would have occurred in thesequence as dictated by, e.g., the template, in the case of enzymaticsynthesis. Similar programmed substitutions can be made using3'-O-phosphoramidites of the individual fluorescent analogs duringstandard phosphotriester synthesis. Thus, for example, the complementarysequence of the Chlamydia tracheomatis MOMP gene, or its fluorescentanalogous sequence, can be synthesized enzymatically using dATP or dFTP,respectively, in the presence of DNA polymerase, dCTP, dTTP, and dGTP:##STR1## wherein the fluorescent deoxyformycin A (F) residues underlinedin the analogous sequence are the structural analogs of thedeoxyadenosine (A) residues in the same relative positions in thecomplementary sequence.

"FRET acceptor" or "Fluorescence Resonance Energy Transfer acceptor"refers to a substance, substituent, chromophore, or fluorophore, e.g., adansyl, naphthyl, anthryl, pyrenyl, methylumbelliferone, or coumarinmoiety, which is capable of absorbing emitted light from fluorescentstructural analog donors and re-emitting that energy at other, longerwavelengths. In the context of the present invention, such secondaryfluorophores may be selectively excited as a second label, or may beused as a fluorescence acceptor to broaden and enhance the primaryfluorescence of the structural analog energy donor.

A. Structures, Sources, Synthesis, and Derivatization of the FluorescentNucleoside Analogs

Briefly, the present invention includes the heterocyclic pyrimidine orpurine structural analogs of the commonly occurring nucleoside bases (B)which are fluorescent under physiological conditions and which arelinked by a carbon-carbon or carbon-nitrogen bond to the set of furanoserings (designated F in FIGS. 4-9) of ribose (R₁₂ ═R₁₄ ═OH), deoxyribose(R₁₂ ═H, R₁₄ ═OH, or R₁₂ ═OH, R₁₄ ═H), or dideoxyribose (R₁₂ ═R₁₄ ═H)and their derivatives such as are described below, and/or are apparentto one familiar with nucleotide chemistry. For the present invention,formycin, 2-amino purine ribonucleoside, and 2,6-diamino ribonucleoside,all of which can (i) form the same or related base-pairing hydrogenbonds as adenosine, and (ii) substitute specifically for adenosine inWatson-Crick base pairing as well as in a wide variety of enzymaticreactions including nucleic acid replication, ligation, andphosphorylation, are used as representatives of the set of fluorescentnucleosides and nucleoside analogs (FIG. 4). Related properties andparallel claims obtain in the present invention for all otherfluorescent analogs of guanosine, cytidine, thymidine, uridine, inosine,and their derivatives.

(1) Structures of the nucleoside analogs. The generic purine andpyrimidine structures of each type of structural analog to the commonlyoccurring nucleosides are given at the top of each of FIGS. 5 through11, below which are representative examples of each class of analog.Only examples of the purine analogs are given in FIGS. 6 and 7, sincethe known pyrimidine analogs have already been illustrated in FIG. 5.With the exception of the N-nucleoside analogs, which have onlysubstitutions at R₄, R₆, and R₉, the generic structures at the top ofeach page show an oval encircling the part of the structure wheresubstitutions to the heterocyclic base distinguish the analog from thecommonly occurring N-nucleosides shown in FIG. 1.

(2) Furanose moieties common to the fluorescent nucleoside analogs. Thenumbering of the sugar carbon atoms in furanose is 1' to 5' as indicatedin FIG. 2; thus the base, B, is connected to C1 of the sugar. Thefuranose moiety of any fluorescent heterocycle claimed in this inventionhas, in common with all other analogs, the set F, of glycosides andsubstituted glycosides, as follows: substitutions can be made, inprinciple, at any of the 5 sugar carbons; the subset F is defined byderivatives and/or substitutions at positions R₁₀, R₁₁, R₁₂, R₁₃, andR₁₄, which (i) are apparent to one skilled in the art, and (ii) are thefuranosyl derivatives of all the fluorescent nucleoside analogs claimedin the present invention. These include all phosphorous substitutions(e.g., triphosphate, thiophosphate, aminophosphate, etc.) and allprotecting substitutions (e.g., dimethoxytrityl) at position R₁₀. Forall glycosides, F, in FIGS. 5 through 11, R₁₀, R₁₁, R₁₂, R₁₃, and R₁₄are defined as follows: R₁₁ and R₁₃ ═H; R₁₄ ═H, OH, or OR_(i) ; R₁₂ andR₁₀ are either H, OH, OR_(m), or NHR_(k), wherein (a) R_(i) protectinggroups are typically lower aryl or alkyl ether, e.g., methyl, t-butyl,benzyl, o-nitrobenzyl, p-nitrobenzyl, o-nitrophenyl, or triphenylmethyl;or a lower alkyl or aryl ester such as acetyl, benzoyl, orp-nitrobenzoyl, or an alkyl; acetal such as tetrahydropyranyl; or asilyl ether, such as trimethylsilyl or t-butyl-dimethylsilyl; or asulfonic acid ester such as p-toluenesulfonyl or methanesulfonyl; orhalide such as bromine, fluorine, or iodine. Additional examples ofsuitable blocking groups may be found in Green, T. W. (1981) ProtectiveGroups in Organic Synthesis, New York: Wiley & Sons. Alternatively, R₁₄may be a FRET derivative including, but not limited to, suchfluorophores as 7- 3-(chlorodimethylsilyl)propoxy!-4-methylcoumarin,O-4-methylcoumarinyl-N- 3-triethoxysilyl)propylcarbamate, andN-3-triethoxysilylpropyl)dansylamide; (b) R_(m) represents anappropriate protecting, substituting, or reactive linker group including2' or 3'-amido, 2' or 3'-azido, 2',3'-unsaturated, and the subset ofphosphorous derivatives involved in chemical or enzymatic syntheses ofoligonucleotides having a phosphate ester, thiophosphate ester, oraminophosphate ester backbone; (c) R_(k) is any common, standardnitrogen protecting group, such as those commonly used in peptidesynthesis (Geiger, R., W. Konig 1981!In The Peptides: Analysis,Synthesis, Biology, Vol. 3, E. Gross, J. Meienhofer, eds., AcademicPress, New York, pp. 149); this includes, but is not limited to,acid-labile protecting groups such as formyl, t-butyloxycarbonyl,benzyloxycarbonyl, 2-chlorobenzyloxycarbonyl, 4-chlorobenzyloxycarbonyl,2,4-dichlorobenzyloxycarbonyl, furfuryloxycarnonyl, t-amyloxycarbonyl,adamantyloxycarbonyl, 2-phenylpropyl-(2)-oxycarbonyl,2-(4-biphenyl)propyl-(2)-oxycarbonyl, triphenylmethyl,p-anisyldiphenylmethyl, di-p-anisyl diphenylmethyl,2-nitrophenylsulfenyl, or diphenylphosphinyl; base labile protectinggroups such as trifluoroacetyl, 9-fluorenylmethyloxycarbonyl,4-toluenesulfonylethyloxycarbonyl, methylsulfonylethyloxycarbonyl, and2-cyano-t-butyloxycarbonyl; as well as others, such as chloroacetyl,acetoacetyl, 2-nitro-benzoyl, dithiasuccinoyl, maleoyl, isonicotinyl,2-bromoethyloxycarbonyl, and 2,2,2-trichloroethyloxycarbonyl;alternatively, R_(k) may also be any reactive group derivatizible with adetectable label (NH₂, SH, ═O, and which can include an optional linkingmoiety including an amide, thioether or disulfide linkage, or acombination thereof with additional variable reactive groups R₁ throughR₃, such as R₁ --(CH₂)_(x) --R₂, where x is an integer in the range of 1and 8, inclusive; and R₁, R₂, and R₃ are H, OH, alkyl, acyl, amide,thioether, or disulfide) or any linker or spacer functioning as ahomobifunctional or heterobifunctional linker including, but not limitedto, such reactive groups as hydrazides, maleimidazoles, oxidizablediols, and succinimydyl groups. At most only one of R₁₂ and R₁₀ may beNHR_(k).

The invention further includes novel phosphoramidites having theformula: ##STR2## wherein B is any of the fluorescent nucleoside analogsdescribed herein and R₁₀, R₁₁, R₁₂, R₁₃ are as defined for the set ofglycosides, F, as above, and R₁₄ may be either H or OH. R₁₆ ═loweralkyl, preferably lower alkyl such as methyl or isopropyl, orheterocyclic, such as morpholino, pyrrolidono, or2,2,6,6-tetramethylpyrrolidono; R₁₅ =methyl, beta-cyanoethyl,p-nitrophenyl, o-chloronitrophenyl, or p-chlorophenyl. All other Rgroups are as before including those identifying spacer or linker armsof from 1 to 25 carbon atoms in length. Prior to the synthesis of thephosphoramidite at R₁₂ in order to (i) preserve any reactivesubstituents on the heterocycle which are important to its participationin Watson-Crick base pairing, and (ii) render the amidite compatiblewith the DNA or RNA chain assembly chemistry, the base moiety B in thephosphoramidite can be protected, which generally involves acylation oramidation of the exocyclic amino groups and includes, but is not limitedto, acetyl, benzoyl, isobutryl, succcinyl, phthaloyl, or p-anisoyl; suchamidine groups include, but are not limited to, dimethylformamidine,di-n-butylformamidine, or dimethylacetamidine; if B is substituted withother reactive groups such as carboxyl, hydroxyl, or mercapto, these areappropriately protected as well.

The present invention encompasses the synthesis of oligonucleotides on asolid phase support, wherein the oligomer is reacted with the protectedfluorescent nucleoside analog phosphoramidites as illustrated in FIGS. 5through 11 and derivatized as in the structure, above. Additionally, thepresent invention includes the novel fluorescent oligonucleotides havingincluded in their sequences at least one fluorescent nucleoside analogderivatized as the phosphoramidite in the structure, above. Moreover, itis yet again another aspect of the present invention to providefluorescent oligonucleotides made by the reactions of the aforementionedfluorescent analog 3'-O-phosphoramidites which are bound to, or havebeen bound by, a solid support.

(3) Sources and other preparations of the fluorescent structuralanalogs. Formycin A is isolated as the ribonucleotide from the culturebroths of Nocardia interforma. The antibiotic is also isolated fromculture broths of Streptomyces lavendulae and Streptomyces gummaensis,and is one of numerous microbial C-ribonucleoside analogs of theN-nucleosides commonly found in RNA from all sources. The othernaturally occurring C-ribonucleosides which have been isolated frommicroorganisms (FIG. 5) include formycin B, oxoformycin B,pseudouridine, showdomycin, pyrazomycin, and minimycin. Formycin A,formycin B, and oxoformycin B are C-nucleosides or pyrazolopyrimidinenucleosides of the class shown in FIG. 6 and are structural analogs ofadenosine, inosine, and hypoxanthine, respectively; a pyrazopyrimidinestructural analog of guanosine obtained from natural sources has notbeen reported in the literature but can be chemically synthesized fromthe 2-chloro-formycin B or its deoxy form. A thorough review of thebiosynthesis of these compounds is available in Ochi et al. (1974) J.Antibiotics xxiv.: 909-916. Synthesis of the N₄ and N₆ derivatives ofthe C-nucleotides are described in Lewis and Townsend ( 1980! J. Am.Chem. Soc. 102: 2817). Corresponding syntheses for the isomericaminopyrazolo- 3,4d!-pyrimidines are in Wierchowski et al. (all othersare commercially available in ribose, and several in deoxy and dideoxyforms, including the azanucleotides and deaza nucleotides, or can besynthesized de novo, e.g., 7-deazaadenine (Gersler et al. 1967! J. Med.Chem. 10: 326)). C-nucleoside analogs of the pyrazolo-s-triazine class(e.g., pyrazolo 1,5a!-1,3,5-triazine) were prepared from aminopyrazole-C-nucleoside as originally described (Fox et al. 1976! J.Heterocycl. Chem. 13: 175).

Production of the deoxy, dideoxy, and phosphorylated forms of thefluorescent ribonucleoside analogs. Chemical syntheses are available inthe literature for the derivatization as 2'-deoxy forms and 3 '-deoxyforms of N-nucleoside, ethenonucleosides as well as the C-nucleosides(Robins et al. 1973! Can. J. Chem. 51: 1313; Jain et al. 1973!J. Org.Chem. 38: 3719; DeClerq et al. 1987! J. Med. Chem. 30: 481). Similarprocedures obtain for the deoxy forms of the azanucleotides,deazanucleotides and are found in the same and additional sources (e.g.,Robins et al. 1977! Can. J. Chem. 55: 1251; DeCierq et al., supra).Protocols and procedures for synthesis of the 3'-azido, 3'amino,2',3'-unsaturated, and 2',3'-dideoxy analogs are as reported (Linet al.1987! J. Med. Chem. 30: 440; Serafinowski, P. 1987! Synthesis 10: 879).Protection or derivatization of the 2'-OH with silyl or FRET moietiescan be done as by Peterson and Anderson ( 1989! Silicon Compounds:Register and Review, Petrarch Systems, Inc., pp. 60-70).

Reported herein is the novel application of a cyclic protectionprocedure from the ribose to the deoxyribose conversion of C-nucleosidesby which only the 2'-deoxy form of the analog is produced, and by meansfrom which high yields can be obtained without the difficultpurification necessary to separate the two isomers produced using theacetoxyisobutyryl halide procedures cited above.

For enzymatic syntheses, mono- and triphosphate forms of the nucleosideanalogs can be prepared by enzymatic phosphorylation with, e.g.,polynucleotide kinase using established procedures, or by chemicalphosphorylation. In general, the 5'-monophosphates are preparedchemically by the POCl₂ (Smith and Khorana 1958! J. Am. Chem. Soc. 80:1141; Yoshikawa et al. 1967! Tetrahedron Lett. 5095). The correspondingtriphosphates can be chemically synthesized according to the sameauthors or Michelson ( 1964! Biochim. Biophys. Acta 91: 1); or Hoard andOtt ( 1965! J. Am. Chem. Soc. 87: 1785). That is, the monophosphates aretreated with carbodiimide (CDI) followed with tributylammoniumpyrophosphate to give the triphosphorylated form. Where it is desired tophosphorylate analogs with exposed amino groups, such substituents canbe thioacetylated by treatment with ethyl trifluorothioacetate accordingto the procedure of Thayer et al. ( 1974! Biochem. J. 139: 609).

B. Synthesis of Fluorescent Oligonucleotides

The present invention presents synthetic methods for the introduction ofone or more of the fluorescent nucleoside analogs of the commonlyoccurring nucleotides into synthetic oligonucleotides.

(1) Use of fluorescent phosphoramidites. Fluorescent phosphoramiditescan be synthesized from the ribose and deoxy-ribose monomers of thefluorescent nucleoside analogs. According to the present invention,fluorescent residues are introduced into chemically synthesizedoligonueleotides by first synthesizing the protected3'-O-phosphoramidite of a nucleoside analog, e.g., 2'-deoxyformycin A;the phosphoramidite is then substituted for the corresponding standardphosphoramidite, in this case deoxy-adenosine-3'-O-phosphoramidite, andreacted with the oligonucleotide being synthesized on a solid supportusing standard phosphotriester chemical synthesis. The β-cyanoethylderivatives may be selectively inserted at any desired position in achemically synthesized oligonucleotide to produce oligomers ofprescribed sequences of 60 or more bases in length and carrying anypredetermined number of fluorescent bases.

For example, non-self-hybridizing oligonucleotides were synthesizedwhich had the perfectly alternating sequences, AC!_(x) and FC!_(x),where x is the number of AC and FC dimer pairs and x had values of x=10,15, 20, 25, 30, gave nearly identical values for both repetitive (>98%)and overall synthesis yields, and produced oligomers which differed onlyin that FC!_(x) was fluorescent, whereas AC!_(x) was not. Both oligomershybridized specifically with complementary alternating oligomers of thesequence TG!_(x) but not with themselves or with noncomplementarysequences such as AG!_(x) and TC!_(x) as indicated by (i) ethidiumbromide staining in agarose gels and (ii) the melting behavior of thehybrids. Equivalent values of the melt transition temperatures in 0.075MNaCl for the FC!_(x) : TG!_(x) and AC!_(x) : TG!_(x) hybrids varied byless than 1° C. for a given value of x (length of oligonucleotide).Specifically, one aspect of the present invention involves the synthesisof 3'-O-phosphoramidites of the fluorescent nucleotides and of theirfluorescent structural analogs, the use of amidites to synthesize highlyfluorescent oligonueleotides having prescribed sequences and the uses ofsuch oligonucleotides as amplification primers, fluorescentoligonucleotide "tags," and hybridization probes.

(2) Use of fluorescent polyribonucleotides and polydeoxyribonucleotides.Fluorescent polyribonucleotides and polydeoxyribonucleotides ofprescribed sequences can be synthesized enzymatically using DNAtemplates from a variety of sources including those prepared by chemicalsynthesis, cloning techniques, or obtained from genomic DNA.Representative syntheses of RNA oligonucleotides using three such DNAtemplates, E. coli RNA polymerase, the rNTPs cytidine, uridine, andguanosine, together with the ribose triphosphate of either formycin A oradenosine, are illustrated in FIG. 12. A representative asymmetricsynthesis of an RNA probe using a template bearing directional viralpromoters, the vital RNA polymerases, the rNTPS cytidine, uridine, andguanosine together with the ribose triphosphate of either formycin A oradenosine, is illustrated in FIG. 13. Symmetric polydeoxyribonucleotideshave been made by substituting 2'-deoxyformycin A-5'-triphosphate (FTP)for deoxyadenosine-triphosphate (dATP) in standard DNA polyerasesyntheses and in DNA amplifications using thermostable DNA polymeraseenzymes and the polymerase chain reaction; the corresponding asymmetricsyntheses have been achieved using the same reagents and procedures butwith the following modifications: (i) syntheses using such DNApolymerase as Klenow fragment or modified T7 DNA polymerase employed atemplate into which a primer site such as the M13 forward primersequence was incorporated into one strand of a duplex at the beginningof the sequence that was to be used as the template, and thecorresponding primer was used to initiate all syntheses; (ii) primerscomplementary to only one strand of a template were used inamplification as is commonly described as asymmetric PCR; or (iii)paired primers in which one of each pair of primers was coupled to alinker such as biotin were used in standard DNA amplifications such asPCR, but one strand was preferentially removed by subsequent isolationsuch as by use of an avidinylated column or magnetic beads. Comparablesyntheses can be made by other substitutions, including, e.g., thefluorescent N-nucleosides, 2-amino purine, and 2,6-amino purine (alsosubstituted for adenosine-5'-triphosphate) and either of the fluorescentC-nucleoside triphospates of formycin B or 5-amino-formycin B(substituted for inosine triphosphate and guanosine-triphosphate,respectively) in either their ribose and deoxyribose forms.

C. Labeling of Fluorescent Polynucleotides

RNA and DNA can be enzymatically labeled by several methods including,but not limited to, (i) 5' DNA end-labeling using both the forwardphosphorylation reaction (Richardson, C. C. 1965! PNAS 54: 158) or theexchange kinase reaction (Van de Sande et al. 1973! Biochemistry 12:5050); (ii) mixed primer labeling by extending mixed sequencehexadeoxynucleotides annealed to restriction fragments (Feinberg, A., B.Vogelstein 1983! Anal. Biochem. 132: 6; Feinberg, A., B. Vogelstein1984!Anal. Biochem. 137: 266); (iii) 3' DNA end-labeling using theenzyme, terminal deoxynucleotidyl transferase, to catalyze therepetitive addition (Okayama et al. 1987! Methods Enzymol. 154: 3;Heidecker, G., J. Messing 1987! Methods Enzymol. 154: 28) ofmononucleotide units of the deoxytriphosphates, or single additions ofdeoxytriphosphates, of several of the fluorescent nucleoside analogs tothe terminal 3'-hydroxyl of DNA initiators, including nonfluorescentprobes of prescribed sequence, e.g., the Chlamydia trachomatis MOMP geneprobe synthesized as described below; (iv) ligase labeling in whichnon-fluorescent "sticky-ended" or "nicked" RNA or DNA oligonucleotidesare labeled by ligation with the appropriate fluorescent RNA or DNAoligomers (Pharmacia LKB 1989! Analects 17.2; Helfman, D. M. 1987!Methods Enzymol. 152: 343); (v) nick translation, in which DNApolymerase is used to incorporate the triphosphates of the fluorescentanalogs randomly in an existing DNA strand in a duplex (Meinkoth, J., G.M. Wahl 1987! Methods Enzymol. 152: 91).

D. Characterization of Fluorescent Oligonucleotides of PrescribedSequences

Hybridization, thermal melting, agarose gel characterization andfluorescence detection studies were used to characterizeoligonucleotides of prescribed sequences. In some cases, the fluorescentoligonucleotides were complementary to known sequences of target DNAfrom clinically important pathogens or mutations, e.g., the MOMP genesequence from Chlamydia trachomatis. In these studies, the templatesused for enzymatic synthesis of the fluorescent oligonueleotides werethe cloned fragments also intended for use later as the target DNA insubsequent hybridization studies. Hybridization of the oligonucleotideswith target DNA results in quenching of the fluorescence of thestructural analogs in a fluorescent probe, which fluorescence isrecovered upon denaturation of the hybrid, thereby proving thathybridization has occurred. The self-hybridization of the syntheticoligonucleotide poly(rFrU), which is discussed at length, below, isrepresentative of the results obtained in such experiments and issummarized in Table 1.

A preferred process according to the subject invention involves fourbasic steps. Initially the fluorescent structural analogs are chemicallyor biologically synthesized and, where appropriate, further derivatizedas required to synthesize a fluorescent oligonucleotide probe. Second, aDNA or RNA probe molecule complementary to a nucleic acid sample ofinterest is constructed to have fluorescent nucleoside analogs which canbe (i) distributed randomly or at specific locations throughout itslength, or (ii) placed as terminal labels as described below. Third, thenucleic acid sample is then separated from unreacted monomers and canthen be characterized directly, used as an extrinsic, non-specific labelfor tagging specific hybridization probes, or used directly as ahybridization probe. In the latter case, hybridization may take place ona solid phase to which either the target DNA/RNA or the fluorescentprobe has been immobilized such as in Southern blot transfers, or"Dot-Blot" techniques, or it may occur in solution (herein, "solutionhybridization"), after which probe/target hybrids are separated fromunhybridized probes by simply washing or filtration. Finally, thefluorescence of the oligonucleotides hybridized to the target DNA/RNA isdetected and quantified.

E. Construction of Fluorescent Probe Molecules

In accordance with the present invention, a preselected fluorescentnucleoside analog or mixture of fluorescent analogs is substitutedspecifically for one or more of the non-fluorescent commonly occurringnucleosides and is then incorporated into DNA or RNA oligonucleotides tocreate prescribed sequences. The prescribed sequences may be chosen tobe equivalent in their Watson-Crick base pairing to a nucleotidesequence constructed from normally occurring nucleotides andcomplementary to a given target DNA or RNA sequence; such fluorescentprobes are said to be analogous to the complementary sequence of thetarget DNA or RNA. The fluorescent probe may be synthesized by eitherenzymatic or chemical synthesis for subsequent applications such as (i)hybridization probes, (ii) amplimers for direct detection of amplifiablegene sequences complementary to a given set of primers, or (iii) asnon-specific "universal" labels which can be attached to specifichybridization probes by, e.g., ligation.

Fluorescent nucleoside analogs of the commonly occurring ribo-, deoxy-,or dideoxyribonucleotides can be incorporated into nucleic acid polymersusing one of several otherwise conventional enzymatic and chemicaltechniques including, but not limited to, those described here.

(1) Enzymatic syntheses. Enzymatic syntheses include:

(a) the use of the enzyme DNase I to introduce small "nicks" into onestrand of a double stranded DNA duplex. The holoenzyme form of E. coliDNA polymerase I can then be used to extend and repair these nicks usinga mixture of fluorescent nucleotide analog triphosphates, e.g.,deoxyformycin-5'-triphosphate (FTP), with commonly occurringdeoxynucleotide triphosphates in the reaction mixture. This methodintroduces a large number of fluorophores randomly throughout the DNApolymer, including both strands of the double helix. In practice, thecommonly occurring nucleotide, in this case dAdenosine-5'-triphosphate(dATP), can be eliminated entirely, and the dFTP substituted in itsplace, without significant loss of synthetic yield, loss ofhybridization specificity, or strength of duplex formation as measuredby the values of the DNA melting temperature;

(b) the use of a variety of enzymes, including the Klenow fragment ofDNA polymerase I and the T4 DNA polymerase, to fill in overhangingsingle stranded regions of DNA produced by the prior actions ofrestriction enzymes. This method concentrates the fluorescent analogs atthe end of each DNA strand. Similarly, fluorescent DNA oligonueleotidescomplementary to a specific DNA template can be synthesized (i) by usingDNA fragments and E. coli DNA polymerase, or (ii) by constructing arecombinant plasmid containing the primer site for a specific primersuch as the M13 forward primer immediately 5' to the desired DNAtemplate sequence. The DNA polymerase will synthesize a complementaryDNA molecule using deoxyribonucleotides or other deoxyanalogs including,e.g., dFTP as a substitute for dATP, present in the reaction mixture;

(e) an incorporation method which also produces a terminal concentrationof fluorescent analogs involves the use of the "tailing" enzyme,terminal deoxynucleotide transferase, to add a homopolymer or "tail" offluorescent deoxy analogs to the 3' end of DNA oligomers. In practice,the yields obtained in the synthesis of homopolymers when substitutingfluorescent analogs for the commonly occurring nucleosides issignificantly less than the yield obtained in the synthesis ofheteropolymers. Alternatively, a single fluorescent nucleoside analogmay be added to the 3' OH of any oligomer using the same enzyme but thedideoxy form of a fluorescent analog or a 2'-protected fluorescentanalog, including the FRET protected analogs, in exactly the same mannerin which, e.g., dideoxy ATP (cordecypin), is used. A third alternativemethod of endlabeling hybridization probes utilizes the action of DNAligase or RNA ligase, by which non-specific double or single strandedfluorescent oligonueleotides can be covalently coupled to either the 3'or 5' end of specific hybridization probes; the fluorescentoligonucleotides used in this fashion do not necessarily participate inthe Watson-Crick base pairing which determines specificity of a probe,but may act solely as a generic or universal fluorescent "tag." Witheach of the foregoing methods, the DNA probes are double stranded andmust be denatured to single stranded form using either heat or alkalitreatment prior to their use for hybridization;

(d) an incorporation method, which can also be used as a standard methodof production of fluorescent probes having a prescribed length andsequence, using standard methods of DNA amplification or replication andone of several available DNA polymerases, including but not limited tothe thermostable DNA polymerases, e.g., Taq polymerase, modified T7 DNApolymerase, Klenow fragment, and T4 DNA polymerase, but substitutes oneof the fluorescent deoxyribonucleotide analogs, e.g., 2'-deoxyformycinA-5-triphosphate or 5-amino-deoxyformycin B-5'-triphosphate for ATP andGTP, respectively, in the mix of nucleotide triphosphates. Thefluorescent oligonucleotides are equivalent in yield and length to thenon-fluorescent oligomer made with the commonly occurring nucleotidesand hybridize to target template DNA and display the same thermalstability and capacity to stain with ethidium bromide as do thenonfluorescent controls once the hybrid duplex has formed. In suchamplifications, the production of fluorescent oligonucleotides can betaken directly as evidence of the presence of a particular sequence, orthe identity can be further established by (i) hybridization with adefined complementary probe, and (ii) sequencing to establish theanalogous sequence; and

(e) the use of fluorescent RNA oligonueleotides complementary to aspecific DNA template which can be synthesized (i) symmetrically, byusing DNA fragments and, e.g., E. coli RNA polymerase as illustrated inFIG. 12, or (ii) asymmetrically, as shown in FIG. 13, by constructing arecombinant plasmid containing the promoter for a specific DNA dependentRNA polymerase immediately 5' to the desired DNA sequence which is usedas a template, e.g., a DNA template bearing a T7 RNA polymerase promoterimmediately 5' to the fragment of a cloned Chlamydia MOMP gene fragmentwhich has the sequence which will be used as the target forhybridization with the probe. For most applications, asymmetricsynthesis is the preferred method, and the corresponding DNA-dependentRNA polymerase will synthesize an RNA molecule using ribonucleotides,e.g., FTP as a substitute for ATP and UTP instead of TTP, which is theanalogous complement to one, and only one, of the two strands of thetemplate. The resulting single stranded probes can be used directly in asubsequent hybridization reaction without a denaturing step.

(2) Chemical syntheses. The protected fluorescent deoxynucleosideanalog-3'-O-phosphoramidites, typically those in which R₁₀═dimethoxytrityl, R₁₆ ═isopropyl, and R₁₅ ═methyl or beta-cyanoethyl,are coupled to the 5'-OH of a growing oligonucleotide attached to asolid support using standard phosphoramidite DNA synthesis techniques(see Atkinson, T., and M. Smith 1984!In Oligonucleotide Synthesis: APractical Approach, M. J. Gait, ed., IRL Press, Oxford, pp. 35-82).Solid support-bound oligonucleotide, which has already been acid washedto deprotect the 5'-OH group, is reacted with 5'-trityl protecteddeoxynucleoside analog-3'-O-phosphoramidite in anhydrous acetonitrile inthe presence of tetrazole under argon, washing away excess reagents, andthen oxidizing the phosphite product to the desired phosphate with asolution of iodine in aqueous THF, and washing with anhydrousacetonitrile. After acid washing to deprotect the new 5' terminus, thecycle can be repeated as many times as necessary to achieve the desiredlength and sequence; additional nucleotides which are added may be thecommonly occurring nucleotides or they may be additional fluorescentnucleoside analogs. Accordingly, one or more fluorophores may beincorporated within a given probe up to and including completesubstitution of, e.g., all of the A residues in a desired sequence withformycin residues. The couplings can be performed manually in aminireactor vial, utilizing a 10 minute coupling time, or on a PharmaciaLKB Gene Assembler or similar instrument utilizing the programmedsynthesis protocols. The fluorescent oligonucleotide is then isolated bycleaving the DNA from the porous glass support by incubation at 55° C.overnight in NH₄ OH:ethanol (3:1). The fluorescent DNA containingammonium hydroxide solution can then be quickly dried in a Speed-Vac andthen separated from failure sequences of a QEAE-HPLC column using ashallow salt and pH gradient. Yields for the nucleoside analogphosphoramidites are comparable to those obtained with standard amiditesbased on repetitive yield calculated from trityl cation release at thedeprotection step.

To provide specific illustrations of how to construct and use probemolecules containing a fluorescent nucleoside analog, following areexamples which illustrate procedures, including the best mode, forpracticing the invention. These examples should not be construed aslimiting. All percentages are by weight and all solvent mixtureproportions are by volume unless otherwise noted.

EXAMPLE 1 Chemical Conversion of Formycin A to 2'-DeoxyFormycin A andPreparation of the 5'-Triphosphate and3'-O-(2-cyanoethyl)-N,N,-Diisopropyl Phosphoramidite

FIG. 16 depicts the invention scheme used to make the2'-deoxy-5'-triphosphate or 2'-deoxy-3'-O-phosphoramidite of formycin A.While the first phase has been previously accomplished by the reactionwith a-acetoxyisobutyryl halides as described by De Clerq et al. ( 1987!J. Med. Chem. 30: 481), the procedure produces both the 3' and 2' deoxyforms which are difficult to separate and are produced in low yield. Thepresent invention employs a 3',5'-disila protection which has previouslybeen applied successfully in the conversion of adenosine to2'-deoxyadenosine ( 1981! J. Am. Chem. Soc. 103: 932). The methodappears to be generally applicable to the corresponding conversion ofmany fluorescent nucleoside analogs.

(I) 7-amino-3-3'5'-O-(1,1,3,3-tetraisopropyl-1,3-disiloxane-dilyl)-β-D-ribofuranosylpyrazolo 4,3d!pyrimidine.1,3-dichloro-1,1,3,3-tetraisopropyl-1,3-disiloxane (0.9 g, 2.85 mMol)was added to a suspension of formycin A which had been exhaustivelydehydrated (0.66 g, 2.5 mMol) in anhydrous pyridine and the reaction wasstirred at room temperature for 24 hours. The solvent was removed undervacuum at T═40° C. and the product extracted between ethyl acetate andwater. The ethyl acetate phase was washed, in seriatim, with (i) cold 1NHCl, H₂ O, aqueous NaHCO₃ (saturated) and aqueous NaCl (saturated)followed by evaporation to a gum. Following flash chromatography onsilica gel and stepwise elution with (i) 2.5% methanol-chloroform, and(ii) 5% methanol-chloroform, the product, which ran as a single spot onsilica TLC (R_(f) =0.80 in 20% methanol-chloroform), was shown to be the3',5' cyclic protected product by proton NMR and elemental analysis.

(II) 7-amino-3-3',5'-O-(1,1,3,3-tetraisopropyl-1,3-disiloxane-dilyl)-2'-(phenoxythiocarbonyl)-β-D-ribofuranosyl!pyrazolo4,3d!pyrimidine. 480 mg of disila protected formycin A (0.93 mMol) wasdissolved with DMAP (0.9 g, 7.6 mMol) in anhydrous MeCN. Followingdropwise addition of 200 mL of phenoxythiocarbonyl chloride through adry syringe mounted in a ground glass joint, the reactants were stirredfor 24 hours at room temperature, after which solvent was removed undervacuum and the product again partitioned between ethyl acetate andwater. The ethyl acetate phase was washed as above, the solventevaporated, and the residue separated on flash chromatography and elutedwith chloroform-MeCN (50/50). Pooled fractions of the desired productwere identified by proton NMR and elemental analysis and subjected to asecond round of production, as below.

(III) 7-amino-3-(2'-deoxy-β-D-ribofuranosyl) pyrazolo 4,3d!pyrimidine(2'-deoxy formycin A). 240 mg of the product obtained from the proceduredescribed in II, above, were added to 12.5 mg (NH₄)₂ SO₄ in a grossexcess of hexamethyldisilazane. The reaction mixture was refluxedat >60° C. overnight. After evaporation under vacuum, the crude trisylylderivative was redissolved in toluene and reacted withazobisisobutyronitrile and tributyl tin hydride by heating under N₂overnight to attain complete reduction. The product was deprotected inTBAF in THF at 80° C. overnight and, after evaporation, fractionatedbetween ethyl acetate and water. The water layer was concentrated andapplied to a Dowex 50W-X8 column equilibrated in water and then elutedwith 15% NH₄ OH. The principal product (R_(f) =0.3 in 20%methanol-chloroform) was shown to be identical to the purified 2'-deoxyformycin A which had been prepared using the method of De Clerq et al.,supra and by proton NMR and elemental analysis.

(IV) 7-amino-3-(2'-deoxy-β-D-ribofuranosyl) pyrazolo4,3d!pyrimidine-5'-triphosphate (2'-deoxyformycin A-5'-triphosphate). 28mg (0.11 mMol) of 2'-deoxyformycin A was added to a glass stoppered testtube and mixed with 0.2 mL of reagent grade acetone and 0.1 ml ofphosphorous oxychloride. The heterogeneous reaction mixture was storedat 4° C. for 24 hours, during which time the solution turned deepyellow. After cooling and addition of 3 ml cold acetone, 6 mMol ofconcentrated NH₄ OH was added rapidly while mixing. After evaporation ofthe acetone, and reduction of the pH to less than 2, the mixture wasrefluxed for 1.5 hours, then diluted and applied directly to Dowex1-formate, from which 2'-deoxyformycin A-MP was eluted with 0.75M formicacid. 2'-deoxyformycin A-MP was converted to the triphosphate by themethod of Yoshikawa et al. ( 1967! Tetrahedron Lett. 5095).

(V) 7-amino-3-(2'-DEOXY-β-D-ribofuranosyl) pyrazolo 4,3d!pyrimidine-3'-O-phosphoramidite (2'-deoxyformycinA-3'-O-phosphoramidite). 2-deoxyformycin A was treated to attain5'-O-protection with DMT and benzoylation of the 7-amino group bystandard procedures. To 0.3 mMol of the product and 25 mg ofdiisopropylammonium tetrazolide in 1.5 ml. of CH₂ Cl₂ was added asolution containing 0.33 mMol ofO-cyanoethyl-N,N,N',N'-tetraisopropylphosphorodiamidite. The mixture wasmixed for 4 hours and partitioned between CH₂ Cl₂ and chilled insaturated NaHCO₃ solution. The CH₂ Cl₂ layer was washed with saturatedNaCl solution, dried (Na₂ SO₄), filtered, and concentrated. Purificationby filtration through a 2" plug of basic alumina in a 25 mm column,eluting with 9:1 CHCl₂ /ET₃ N, provided the phosphoramidite which couldbe dried to a foam. Identity of the product was verified by proton NMR,elemental analysis, fluorescence of the heterocycle, and use inoligonucleotide synthesis.

EXAMPLE 2 Complete Enzymatic Substitution of FTP or 2'dFTP for ATP ordATP in RNA or DNA Probes

A. Symmetric synthesis of ribose oligomers. RNA oligonucleotides weresynthesized from three DNA templates (FIG. 12) using (i) FTP (F₁₀₅) as asubstitute for ATP, and (ii) a purified E. coli RNA polymerase asoriginally described by Ward et al. ( 1969! J. Biol. Chem. 12: 3242),except that synthesis was allowed to run for three hours at 37° C.before the reaction was stopped; FTP effectively replaced ATP but notany of the other three normal nucleotides CTP, UTP, or GTP.

At the end of the synthesis, reaction products were separated fromunreacted reagents by separation at 4° C. on Sephadex G-50 in normalsaline at pH 7. The scheme for separation of reaction products fromunreacted agents is shown as a flow chart in FIG. 19.

In the reaction, FTP is an effective substrate for RNA polymerase withboth native and denatured DNA as well as with synthetic deoxynucleotidepolymer templates. In samples containing CTP, UTP, GTP, RNA polymerase,one of the DNA templates, and either FTP or ATP, a high molecular weightproduct eluted from either sample in the void volume while the amount ofmonomeric NTP in the retained fraction from either sample wascorrespondingly reduced by >70%. No high molecular weight fraction otherthan the small amount of template eluted from enzyme-free controls andunreacted rNTPs were undiminished; similarly, template-free controlscontained only unreacted rNTPs which co-eluted in the retained volumewith standard ribonucleotide triphosphates. Similar results wereobtained with a variety of DNA templates from natural and syntheticsources, including the alternating copolymers poly d(AC), poly (AG), andpoly (ACGT). Moreover, comparable yields of high molecular weightoligomer were obtained from syntheses in which (i) the N-nucleosideanalogs 2,6-diamino-adenosine-5'-triphosphate or2-diamino-adenosine-5'-triphosphate were substituted for ATP in thereaction mix, or (ii) the C-nucleosides formycin B-5'-triphosphate(F_(b) TP) or -amino-formycin B-5'-triphosphate (aF_(b) TP) weresubstituted for GTP in the reaction mix and using poly (TG) or poly (GC)as the DNA template. No matter what the template, yields obtained bysubstituting several of the deaza- and aza-nucleoside analogs for ATP orGTP were dramatically lower.

B. Asymmetric synthesis of RNA or DNA probes. In vitro, DNA dependent,RNA polymerase transcription systems for the synthesis of RNAs for useas substrates and hybridization probes are a fairly common tool ofmolecular biology. They are uniquely applied here to the development ofautofluorescent probes and their production. The method developed isgeneral and applies to any of the phage polymerase systems, includingSP6, T7, and T3. In the present case, the invention employs a pair ofpromoters which are separately positioned on alternate strands of aduplex plasmid and at opposite ends of a polylinker as shown in FIG. 13.The vectors are used to (i) attach promoters capable of effectingasymmetric synthesis through use of a viral polymerase which recognizesone of the promoters, and (ii) replicate multiple copies of a templatefor use in asymmetric production of a fluorescent probe or of anonfluorescent copy of the probe target. A copy of the DNA targetsequence is inserted into the polylinker in its duplex form and at arestriction site adjacent to one of the promoters. Replication of theplasmid in competent cells provides large amounts of the template fortranscription. Two separate but parallel methods have been developed forthe asymmetric synthesis of DNA probes. In the first case, ssDNA probesare synthesized from templates which have primer binding site attachedat the 5' end of one template strand as shown in FIG. 14. In suchsyntheses, the primer may be non-fluorescent or may be synthesized usingfluorescent analog phosphoramidites as shown at the fight of the Figure.A variation on this is asymmetric amplification and separation in whichboth strands of a template may be replicated by amplification asfluorescent oligomers, but using a pair of primers in which one, andonly one, bears a transient affinity linker such as biotin which maysubsequently be used to separate the denatured sense and antisensestrands.

For both RNA and DNA probes, it has proven practical to establish areference template, probe sequence, and target sequence against whichall transcriptions and probe detection sensitivities are calibrated. Thealpha chain of Xenopus translation elongation factor (Xef-1α) servesthat purpose and asymmetric RNA probe synthesis is used here asrepresentative of all RNA and DNA synthesis. The Xef-1α mRNA is a majortranscription product of the Xenopus embryo which comprises a largepercentage of the non-mitochondrial mRNA transcripts that appearimmediately after the midblastula transition. The gene for the Xef-1αwas isolated and EcoRI linker sites added at the ends of the cloneduring construction of the cDNA library. The 1705 nucleotide fragmentwas inserted into a pSP72 plasmid bearing a T7 promoter on one strandand an SP6 promoter on the complement. Following plasmid replication andtemplate linearization, transcription with T7 RNA polymerase, the rNTPscytidine, uridine, and guanosine, together with the ribose triphosphateof either formycin A or adenosine, produced 1749-base-long oligomerscontaining 489 F or A residues, respectively. Transcripts less than fulllength were never observed and, in each case, the analogous and controloligomers were produced in comparable quantities and were generallyindistinguishable in physical behavior save that the analogous sequencewas permanently fluorescent.

There are two unique features of this novel manufacturing system. (1)Synthesis of the antisense strand, e.g., using SP6 and the commonlyoccurring nonfluorescent rNTPs provides standardized target sequences inhigh yield. In the corresponding asymmetric synthesis of DNA probes,distinct primer sites on complementary template strands can be used toachieve the same objective. (2) A mixture of plasmids containing severaldifferent plasmids can be used to create a "cocktail" of linearizedtemplates from which the corresponding "cocktail" of probes (see Example7, below), which can bind to multiple sites on a genomic sequence, canbe concurrently transcribed.

EXAMPLE 3 The Fluorescence of Nucleoside Analog RNA Probes and Proof ofTheir Hybridization in Solution

The effective utilization of FTP in the poly d(AT) directed synthesis inExample 1 produced a polymer approximately 300-500 bases in lengthwhich, when hydrolyzed and/or sequenced, proved to be a perfectlyalternating replicate of the DNA template, but with the sequence: poly(FU). As predicted from this sequence, the product could be annealed tolike chains by a single thermal cycle, thereby creating the putativeproduct poly (FU):poly (FU); unlike the comparably treated poly (FC),which showed no evidence of self-hybridization as expected, the annealedhybrids of poly (FU):poly (FU) stained with ethidium bromide in agarosegels and gave a sharp thermal transition in both absorbance andfluorescence, proving that the probes could hybridize both effectivelyand specifically. The absorbance and emission spectra of the purifiedpoly (FU), poly (FC), poly (FG), poly (UF_(b)), poly (CaF_(b)), and poly(FCGU) differ from those of purified poly (AU), poly (AC), poly (AG),poly (TG), and poly (ACGT) controls in four respects: (i) the far UVabsorbance maximum is shifted slightly for the analog-containingproducts, to 265 nm as compared to 260 nm for the controls; (ii) thereis a significant, highly structured absorbance (3 peaks at roomtemperature) between 290 nm and 320 nm with negligible absorbance at 340nm; (iii) an excitation maximum appears at 303 nm; and (iv) there is abroad emission band extending into the visible wavelengths with a peakat 405 nm (Stokes shift =102 nm). It is an important property that thefluorescence is fully quenched in, e.g., the poly (FU):poly (FU) hybrid,and cannot be detected until the strands are denatured by raising the pHof the solution to values >pH 10. Once denatured, the fluorescence ofthe oligomer is fully integratable, with relative fluorescenceintensity >40% of peak intensity over the range 360 nm to 460 nm.

                                      TABLE 1                                     __________________________________________________________________________    Properties of hybrid formation by poly (AU) and poly (FU)                                                INTACT HYBRID                                            DENATURED HYBRID            ETHIDIUM                                    RNA:RNA                                                                             WAVELENGTH MAXIMA    LENGTH BROMIDE                                                                             MELT                                  HYBRID                                                                              ABSORBANCE                                                                            EXCITATION                                                                           EMISSION                                                                            (BASE PAIRS)                                                                         STAINING                                                                            TEMP                                  __________________________________________________________________________    r AU!:r AU!                                                                         260 nm  --     --    150-300                                                                              yes   32° C.                         r FU!:r FU!                                                                         266 nm  303 nm 405 nm                                                                              150-300                                                                              yes   33° C.                         __________________________________________________________________________

EXAMPLE 4 Hybridization of Fluorescent Probes to Target RNAs and TargetDNAs; Uses of Linkers to Allow Solid Phase Detection

The synthetic template poly (TG) was used to produce the complementaryRNA probes poly (AC) and poly (FC), neither of which is selfcomplementary and in which hybrids could not be annealed or detected; ofthe two only the poly (FC) was fluorescent. In a parallel experiment, apoly (AC) template was amplified using the biotinylated synthetic 22-merprimers, ⁵α BIOTIN-(TG)₁₁ ^(3'), together with standard polymerase chainreaction (PCR) methods to produce the DNA amplimers having the sequence,⁵α BIOTIN-poly (TG)^(3'), then separated from the unreacted primers bygel sizing and/or QEAE ion exchange chromatography, after which thepolymers were radioactively labeled using ³² P-ATP and the enzymepolynucleotide kinase. When mixed separately, but in equimolar amounts,with the biotinylated amplimers, ⁵α BIOTIN-poly (TG)^(3'), both of theRNA probes, poly (AC) and poly (FC), formed hybrids which could becharacterized by (i) ethidium bromide staining, and (ii) meltingbehavior; as expected, the fluorescence of the poly (FC) probe wasquenched by hybridization. The hybrids could then be adsorbed via the^(5') BIOTIN moiety to avidinylated beads, washed to remove unhybridizedpoly (FC), and equal aliquots assayed for radioactivity andfluorescence. Prior to denaturation of the washed sample, detectablefluorescence in the solution was negligible; when denatured in high pHbuffer, the amount of poly (FC) which had been hybridized, whenestimated from the fluorescence of standardized dilutions of the probe,was within 1% of the amount of the target DNA, ⁵α BIOTIN-poly (TG)^(3'),as measured by the amount of radioactive label in the sample as comparedto standardized dilutions.

EXAMPLE 5 Hybridization of Fluorescent Probes Synthesized fromNucleoside Analog-3'-O-Phosphoramidites to Target DNAs

In a validation of the use of the phosphoramidites of the fluorescentnucleoside analogs, n-mers which varied in length in multiples of 5bases from 25-mers to 60-mers, and having the sequence (AC)_(x) or(FC)_(x), where x=12.5, 15, 17.5, 20, 22.5, 25, 27.5, or 30, weresynthesized in parallel using either dAdenosine-3'-O-phosphoramidite ordF-3 '-O-phosphoramidite together with dC-3'-O-phosphoramidite in aPharmacia LKB Gene Assembler. After cleavage from the solid phase andpurification of QEAE-Sepharose, the fluorescent oligomers (FC)_(x) ofdefined length could be hybridized to the radiolabeled amplimers of poly(TG), from Examples 2 and 3, above, as assessed by DNA melting behavior,ethidium bromide staining, and the reappearance if quenched fluorescencefollowing denaturation of the hybrid.

EXAMPLE 6 Assay for Chlamydia trachomatis Using an FTP Substituted RNAProbe

Chlamydia trachomatis is an obligatory intracellular pathogen which, inits active infectious stages, contains from 3×10³ to 4×10³ copies ofribosomal RNA (rRNA) and one copy of genomic DNA/bacterium. A primerpair, one of which contained a 5'-biotinylated T7 promoter which was 5'to the hybridizing primer sequence, was used to amplify a 150 base pairDNA segment of the MOMP gene from a stock strain of C. trachomatis L2.Approximately 500 ng of the DNA fragment, which contained the T7 RNApolymerase promoter at the 5' end, was transcribed with T7 RNApolymerase in the presence of rCTP, rUTP, rGTP, and with either rFTP orrATP (+control). The reaction was stopped by heat inactivating theenzyme for 3 minutes at 100° C. Unincorporated rNTPs were separated fromthe labeled RNA by gel sizing chromatography on a Sephadex G-25 column,after which the probe concentration was estimated from its absorbance at260 nm. Using a simple dual monochromator fluorescencespectrophotometer, as little as 5×10⁻¹⁴ moles of the RNA probe could bedetected over background when 20 nm slits were used for both excitationand emission monochromators. A photon counting fluorimeter designed forsensitivity (see Example 9, below) is capable of detecting between5×10⁻⁶ and 5×10⁻¹⁷ moles of the same probe, equivalent to the amount ofribosomal RNA expected from between 5000 to 50,000 of .the bacteria. Twohundred microliters of either (i) C. trachomatis genomic DNA, or (ii)the amplified target DNA were mixed with 200 μL of a 1/200 dilution ofthe probe in hybridization buffer (0.15M NaCl, 0.02M sodium citrate,0.02M HEPES, 0.004M EDTA, pH 7.4) and the mixture boiled for 3 minutes,after which they were allowed to cool slowly to room temperature overone hour. An aliquot of the genomic DNA sample was eluted into anultrafiltration microtube or 96-well filter plate (pore size=0.1 μm) asillustrated in FIG. 17, washed 5 times with 0.15M NaCl, 0.02M sodiumcitrate, pH 7.4, after which the sample was divided in two, one halfdenatured in high pH buffer, and both aliquots scanned to measurefluorescence background and the fluorescence of hybridized probe,respectively. Target DNA amplimers were treated similarly except thatthe 5'-biotinylated primer end of the target DNA segments were firstadsorbed to avidinylated magnetic beads (2.8 μm diameter) so that thesample could be washed without loss of material (FIG. 18). With eithertreatment, fluorescence of the probe may be detected at dilutions of thesample which contain less than 1×10⁻⁶ moles of target DNA, which isroughly equivalent to the sensitivity required to detect less than10,000 bacteria if a single similarly sized probe were used to detectrRNA from infectious Chlamydia. The probe used here is about 150 basesin length, contains approximately 38 formycin residues per probe, andbinds only to a single target site on each copy of the ribosomal RNA. Itis an important feature of this invention that increasing the number offluorophores in a probe, or probe "cocktail," also increases thesensitivity of detection. With 13 times as many formycin residues perprobe as the 150 base MOMP gene probe, 1×10⁻¹⁸ moles of the Xef-1α probecan be detected in a dual monochromator fluorescence spectrophotometerwhereas less than 1×10⁻²⁰ moles are detected using the photon countingtechnology described in Example 9.

EXAMPLE 7 Detection of Multiple Target Sites

An important aspect of the asymmetric syntheses to both diagnostic andtherapeutic, e.g., antisense, applications of nucleic acid probes is thecapacity for concurrent synthesis of probe "cocktails" which maycomprise probes which differ in length or differ in the locations ornumbers of the target sites on RNA or genomic DNA to which they willbind. Utilization of probe cocktails to three different types ofdiagnostic targets illustrate the broad importance of this feature.

A. Single target nucleic acids present in multiple copies. In somespecies of pathogen, multiple copies of rRNA are present in eachorganism, e.g., each bacterium of Chlamydia trachomatis containsapproximately 2×10⁴ rRNA molecules per organism. Since the rRNA ofChlamydia is typically between 3000 and 5000 nucleotides in length,sensitivity in a diagnostic assay may be increased significantly by useof a probe cocktail specific for target sequences on rRNA and made of asmany as 5 to 10 different probe sequences, each of which can bind todiscrete segments of the target rRNA or target DNA as indicated withprobes (a) to (e) in the lower half of the diagram shown in FIG. 20 inwhich (a), (b), (c), (d), and (e) are analogous complementary probesspecific for different target sequences of a single DNA strand.

There are two disadvantages in using rRNA sequences as diagnostictargets: (i) rRNA sequences are highly conserved, hence only shortvariable sequences are useful for the detection and identification ofinfectious pathogens. One consequence of this to diagnostic sensitivityis that only limited numbers of `reporter` labels can be used on eachprobe, thereby limiting sensitivity; and (ii) only a few pathogens carryrRNA in high copy numbers, and many, such as the DNA viruses, carry norRNA at all, hence the number of diagnostics which can employ thisstrategy is limited.

B. Multiple different target sequences on a single strand of DNA. Thegenomes of all organisms are significantly larger than rRNA andtypically carry more numerous and larger unique segments which can serveas target sequences for nucleic acid probe hybridization. For example,the complete genome of Chlamydia trachomatis has been isolated andconsists of a relative small double stranded DNA with a molecular weightof >660×10⁶ or slightly more than 1×10⁶ base pairs. Each bacterium alsocontains a 4.4×10⁶ dalton plasmid containing >7 kbases. Unlike the rRNAof this species, the plasmid is unique to Chlamydia in its entirety--nocross-hybridization can be detected with the DNA from, e.g., Neisseriagonorrhea--indeed, no cross-hybridization occurs between the differentrestriction fragments of the plasmid itself. Even when no other portionof the Chlamydia genomic DNA is chosen for use as hybridization targets,a cocktail specific for the multiple restriction fragments of theChlamydia plasmid alone is equivalent in length to more than 4 Xef-1αprobes and can be detected at levels equivalent to between 100 and 1000bacteria.

C. Multiple copies of a single target sequence on a single strand ofDNA. It has only recently been discovered that flanking sequences oneach side of several genes contain moderate to long stretches of tandemrepeats. Ribosomal gene repeats are of particular interest in the kindsof DNA based diagnosis described in this invention. Like the ribosomalgenes, they are present in high copy numbers, which improves sensitivityof detection but, in addition, the spacer regions between genes arenormally highly variable from species to species, since they are notsubject to selective pressures. Multiple copies of the same uniquesequence on a single DNA strand represents a special case in which thehybridization targets are a cocktail of loci on each genome; that is, asingle probe sequence can probe multiple target sites of the samesequence and on the same DNA strand. They are ideally suited as speciesand genus specific probe targets.

A representative example of such probes and targets was created for thedifferent species of the protozoan parasite Eimeria, which causescoccidiosis in a variety of domestic animals. Genomic DNA from E.tenella was digested with several different restriction enzymes, and thefragments ligated into appropriately cut asymmetric plasmid vectors andwere used to transform Escherichia coli. Colonies were screened forrepeat sequences by hybridization with Eimeria tenella genomic DNA thathad been labeled with ass by random priming. Strongly hybridizing cloneswere picked and subjected to differential screening with labeled genomicDNA from E. mitis, E. maxima, E. acervulina, and E. tenella, as well asDNA from the closely related genera Plasmodium, Trypanosoma, andSarcocystis. The majority of clones gave signals of equal intensity withDNA from the other genera. Some clones, however, were recognizedspecifically by the Eimeria and one done was recognized only by E.tenella.

The entire sequence of the insert in the latter done contains 334 basepairs. Physical characterization of the restriction fragments indicatesthat the sequence is present in tandemly repeated units of approximately738 base pairs and that a minimum of 30 genes are tandemly linked andall appear to be on one chromosome. Asymmetric probes synthesized usingthe tandem repeat as a template contain 179 formycin A residues pertemplate sequence.

Even when no other portion of the Eimeria genomic DNA is chosen for useas a hybridization target, a single sequence probe specific for only themultiple copies of the tandem repeat on the Eimeria genome is equivalentin length to more than 11 Xef-1α probes. Since the infectious particlesfor Eimeria are oocysts, each of which contains 8 genomes, such cocktailof targets makes it possible to detect less than 10 oocysts. The importof tandem repeat targets extends well beyond sensitivity, however, orsimply the detection of this single genus, since tandem repeat sequencesappear in a genomic DNA of a wide variety of species and genera, and aredistinct for those species, thereby providing a broad basis for thedesign of diagnostic assays for a wide variety of pathogens, includingthose for which no rRNA targets exist.

EXAMPLE 8 The Use of Non-Specific and Non-Hybridizing FluorescentOligomers as Universal Fluorescent "Tags" by Ligation or ChemicalLinkage

Simple modification of the template to produce a "sticky end" at the 3',5', or both 3' and 5' termini, e.g., to 5'ACGT-polyd(AT),polyd(AT)-TGCA^(a'), or ^(5') ACGT-polyd(AT)-TGCA^(3'), respectively,enabled synthesis of nucleic acid probes with all of the aboveproperties, but which could also be ligated, either (i) to like strandsto produce longer fluorescent probes, or (ii) to other hybridizationsequences specific for a prescribed target DNA. The latter is aparticularly useful way in which to produce a universal label for anycloned DNA fragment, and allows a given probe to be identified by twonon-hybridizing but highly fluorescent sequences at its termini, withoutthe need to denature the hybrid for detection as was seen with thesimple poly (FU) probe, above. Equivalent non-hybridizing universalprobes can be readily made by chemical synthesis using, e.g., the ethenoanalog phosphoramidites, e.g., 1,N₆ -ethenoAdenosine-Y-O-phosphoramidite(eA), to synthesize non-specific tags which can subsequently be linkedto any hybridization probe. The 3' or 5' termini of such universalprobes can also be prepared for chemical, rather than enzymaticattachment to other oligomers or solid phases, through the addition of,e.g., 5'-amino hexyl, 5'-sulfhydryl hexyl, 3'-aminohexyl amino,N-hydroxysuccinimide esters, and other such linkers. The uniqueapplication of this probe technology, which employs the universal endlabel, is quantitative and works well for routine assays which requirehigh sensitivity. Another application of this technology referred toherein as "sustained signal amplification" is non-quantitative and canbe useful for a situation where extreme sensitivity is required toanswer "yes" or "no" whether a particular gene marker is at all present,for example, where low copy numbers of a target sequence are present."Sustained Signal Amplification" is described in more detail in Example8(B), below.

A. The 5' Universal End Label

Homopolymers of non-hydrogen bonding fluorescent nucleoside analogs,e.g., ethenoadenosine, can be used together with asymmetric synthesis ofssDNA and RNA to increase the density of fluorescent labeling oncocktails of small probes, on small fragments as in sequencing, and toincrease sensitivity of labeling of small or low copy number target. Thegeneral concept comprises an oligomeric probe constructed along atypical phosphodiester backbone, but which can be divided into distinctfunctional regions-the 5' fluorescent homopolymer; primer, or promotercomplement; an optional "tether" region, which can connect thehomopolymer to the primer; and a target complement. A diagram of thisdescribed 5' universal end label is shown in FIG. 22.

The functional regions of the phosphodiester chain as shown in FIG. 22are:

A=a non-base pairing homopolymer of from 1 to about 50 fluorescentnucleotide analogs;

B=an optional non-nucleotide phosphodiester "tether" comprising, e.g.,one or more freely rotating alkyl chains inserted as part of thephosphodiester backbone of the oligomer;

C=an enzymatic synthesis primer for use in initiating enzymaticsynthesis of the target-specific D region. Representative examples wouldbe the complementary sequences to the T7 RNA polymerase promoter or tothe M13 forwared primer as are used in asymmetric RNA or DNA probesysthesis described herein;

Regions A, B, and C, typically from 20 to 60 bases in length, can bechemically synthesized. The 5' universal end label comprises at leastregions A and C and, alternativley, can also include the optional regionB.

D=a target complementary sequence of from 40 to 20,000 nucleotides inlength. This sequence may or may not include fluorescent nucleotideanalogs, but functions primarily as the region which establishes targetspecificity. This region is also distinct from the other regions in thatit is enzymatically synthesized from templates adjacent to the promoteror primer site to which region C is complementary. The entire 5'universal end label can be used as the primer for DNA or RNA replicationof the target-specific complement.

Enzymatic synthesis using a 5' universal end label is illustrated usingthe M. tuberculosis IS6110 template (a sequence unique to the bacterium)which has been inserted into a standard Gemini plasmid to create asynthesis template. Other plasmids can be used as well. This enzymaticsynthesis process is shown in FIG. 23. The following advantageousproperties of the 5'0 universal end label have also been discovered.

(1) As shown in FIG. 24, the excitation spectrum of one non-hydrogenbonding fluorescent analog, ethenoadesosine (designated F₁₈₅), iscompared with the comparable excitation spectrum of formycin (F₁₀₅). TheF₁₀₅ extends further into the UV wavelengths. Two important discoverieshave been made about both the excitation and emission spectra of F₁₈₅ :(i) the wavelength maxima are the same at both pH 7 and pH 11, and (ii)the quantum yield is more than 10x that of F₁₀₅ having values of 0.55and 0.65 at pH 7 and 11, respectively. This allows the use of the 5'universal end label under a wider variety of pH conditions and canresult in significantly greater luminescence from fewer totalfluorophores. It has been shown that an F₁₈₅ 20-mer which is excited atpH 11 over the range 270 nm≦λ≦310 nm can be equivalent to labeling withbetween 3 and 10 fluorescein molecules. Furthermore, the fluorescencedoes not quench and can be used with time resolved spectroscopy.

(2) non-base pairing end labels do not interfere with primer-mediatedDNA amplification or replication, are water soluble at concentrations upto 10⁻³ M, and do not increase the background in a binding assay due tonon-specific hybridization to non-target sequences.

(3) such probes are beyond the capacity of chemically synthesized probesbecause, as is well known in the art, the practical limit of synthesisin reasonable yield remains approximately 60 bases. The 5' universal endlabel can be used to increase sensitivity of detection by using acocktail of relatively short probes for which the length of the "D"region is approximately 100 bases. For example, as illustrated in FIG.25, the 1361 bp IS6110 sequence of M. tuberculosis has been used as atarget for a cocktail of 10 probes, each having a different "D" segmentor complementary target sequence. Each probe, however, bears the same 5'universal end label. In the case of M. tuberculosis, there are 16 copiesof the IS6110 gene per bacterium. By using the end label in a mannershown in FIG. 25, each bacterium has the potential for being labeled bya probe cocktail with permanent fluorophores which are equivalent ininstantaneous emission to between 480 and 1600 fluorescein molecules.

(4) the same labeling device can be used to provide a standardizedfluorescent label in standard DNA sequencing, but having pre-labeled DNAfragments so that sequences can be read or recorded directly from thegel. Such a use is depicted in FIG. 26.

B. Sustained Signal Amplification (SSA)

The 5' universal end label should prove particularly useful for thosesituations in which a unique genetic marker is present only in arelatively few copy numbers of target genes present in a large genome.For such applications in which exquisitely sensitive levels of detectionare required, or for which very little target is present, somecombination of fluorescent labeling and signal replication oramplification is required. Hepatitis B presents such a case. The entiregenome of Hepatitis B virus (HBV) is only 3200 bases long and, in thevirion, one of the strands is even shorter. The virion contains a DNApolymerase which utilizes nucleotide triphosphates from a host cell tocomplete the short chain as the first step in an infection.

The DNA polymerase of the virion utilized together with a novelfluorescent nucleoside analog described herein was combined with anon-PCR type of amplification which has heretofore been used only forRNA replication. In this scheme, shown in FIG. 27, the virion DNA servesas an in situ template and, in combination with the above-describedasymmetric synthesis method, can be used to amplify the intensity of thefluorescent signal. The process, which may be better understood byreferring to FIG. 27, involves two steps. First, the sample DNA iscombined with (i) deoxynucleotide triphosphates (deoxyNTPs) includingtriphosphorylated fluorescent nucleoside analogs, and, (ii) two primers(shown as A and B in FIG. 27), the first of which has at its 5' end asequence complementary to an RNA polymerase promoter. The primersreferred to in this Example are described as "A" and "B" to indicate theuse of two separate primer. These are described as such for illustrativepurposes and, as such, would be understood by those in the art to referto any primer which comprises a sequence complementary to a promoterregion on the target sequence and which can be used with a nucleic acidpolymerase., In the illustration shown in FIG. 27, the T7 RNA polymerasepromoter is designated by the thicker line at the end of primer A. Thesample is first incubated at 37° C. for 10 minutes to allow the viralDNA polymerase to complete the short genomic strand; the sample is thenraised to 65° C. for 1 minute to denature the genome, after which theprimers are annealed at 42° C. Second, the two enzymes, reversetranscriptase and T7 RNA polymerase, are added, together with theriboseNTPs including the fluorescent ribonucleoside analogs, and theentire sample is incubated at 42° C. for 1 hour. This creates a cyclingsynthesis of DNA strands and RNA strands as indicated in the lower halfof FIG. 27. The net effect is to produce somewhere between 10⁸ and 10⁹fluorescent RNA strands and about 100-fold less fluorescent DNA strands.Following a wash of the sample in a cell-free unit to remove the unusedmonomeric fluorescent NTP's, the sample can be simply read forfluorescence to determine whether any template, in this case Hepatitis BDNA, was present in the sample.

EXAMPLE 9 Quantitation of Luminescent Probe Using Time ResolvedFluorometry

A novel method for detecting fluorescent nucleoside analogs, fluorescentoligonucleotides or analogous sequences, of the amount of boundfluorescent oligonucleotide probe has been developed based on the use ofphoton counting to measure the amount of a fluorophore in a sample andis described herein below. The method differs from time resolvedspectroscopy in that the method integrates all fluorescence emissionfrom a fluorophore or nucleic acid probe, independent of the wavelengthof the emission and is both a novel combination of time and spectralintegration and a novel application of photon counting to theidentification, detection, and quantitation of nucleic acid targetsequences to diagnostic assays and therapeutic treatments.

The fundamental experimental parameter used in any measurement ofluminescence is the intensity of the luminescence, 1, the units of whichare moles of photons per second per liter. Because the fluorescentnucleoside analogs used here are, for all practical purposes,permanently fluorescent and do not photobleach within the lifetime of atypical measurement, the luminescence of fluorescence, measured in molesof photons emitted per second per mole of fluorophore, can be used as anindex of the amount of fluorophore, and hence probe, in a sample. Thepreferred instrumentation for such measurements, developed at Chromagen,comprises (i) a 150 watt Hg/Xe CW cylindrical lamp capable of highintensity excitation over the range 290 nm ≦λ≦320 nm, (ii) an ultrahighsensitivity photomultiplier in which the photodynode is coated to allowa response only over the range of emission 360 nm <λ<550 nm, (iii) acylindrical cuvette with quartz excitation windows but glass walls whichcan serve as the emission filter. The cuvette is mounted so that theentire sample can be collected at the face of the photomultiplier tube,and (iv) 5 computer-driven photon counting clocks, connected inseriatim, and each capable of discriminating between photons at afrequency of 10⁹ per second.

In experiments with the monomeric formycin A and full-length Xef-1αprobe containing 489 formycin residues under conditions of roomtemperature and pH=10, we have found that (i) the luminescence of serialdilutions of the monomer and the probe are linearly related to theconcentration, and (ii) the luminescence of the probe is equivalent tothe same number of free monomers. In a typical assay using permanentfluorophores such as those shown in FIGS. 17 and 18, the amount oftarget present in a sample is determined by denaturing hybrids afterunbound probe has been washed away and measuring the mount of probewhich was bound. The fluorescence equivalence of residues in ananalogous probe sequence to the emission of the same number of monomers,under alkaline conditions used here, indicates that there is negligibleself-quenching in the oligomer and demonstrates that the luminescence ofthe probe can be used directly to quantitate the amount of probe boundby target RNA or DNA, thereby providing a broad basis for the design ofdiagnostic detectors for a wide variety of nucleic acid assays anddiagnostics. It is an important consequence of the invention, thatsensitivity and signal-to-noise ratios are a function of the number ofthe photons counted and the number of time periods over which countingis done.

EXAMPLE 10 Attachment of 5' and 3' Linkers for Immobilization of theOligonucleotides and Hybrids or for Attachment of Fluorescent Oligomersas "Labels"

The chemistries and procedures of the invention can be used to createand characterize any probe synthesized using fluorescent nucleosideanalogs, whether the synthesis is enzymatic or chemical, for bothfluorescence and hybridization specificity. Such probes can be used notonly in the solution hybridization formats described here, but also inthe more frequently used laboratory procedures such as "dot-blot"detection, electrophoresis in agarose or polyacrylamide gels, Southernblotting, and hybridization on filters and membranes, as well asseparation of the hybrids by HPLC or capillary electrophoresis methods.Although linkers are not essential to the solution hybridization, anyappropriate affinity linker such as biotin/avidin or homo- orheterobifunctional linker can be used to capture the probe or hybrid forpurposes of concentration, isolation, or detection, as illustrated forthe PCR amplified DNA fragments of FIG. 18. The present inventionincludes linker derivatized fluorescent nucleotides, as well asoligonucleotides, linker derivatized primers for use in amplificationand subsequent detection with fluorescent oligonucleotide probes,oligonucleotide probes, plasmids, and therapeutics made or otherwise"tagged" therefrom, and/or their uses and applications such as aredescribed herein. Such derivatizations include, but are not limited to,transaminations to purine or pyrimidine nucleosides and/or theirfluorescent structural analogs, amino-thiol, azido-, aldehyde,hydroxysuccinimide, 5' aminoalkyl-3'-O-phosphoramidite,5'-thioalkyl-3'-O-phosphoramidite, 3'-aminohexyl amino, amino silanes,and aminosilyl derivatives and other such linkers and groups reactivewith linkers or in condensation reactions such as Schiff basecondensations of 3' or 5' oxidized cis-diols, as are familiar to oneskilled in the art. To illustrate this a specific case is offered:

(i) a set of non-fluorescent amplification primers for the MOMP genesequence was chemically synthesized; at the end of synthesis anadditional cycle was used to add 5'-aminohexyl-3'-O-phosphoramidite tothe 5' terminus of the completed primer with the addition chemicallysynthesized, using standard phosphotriester chemistry.

(ii) Following cleavage from the solid phase support in strong ethanolicbase, the terminal amino group, of each strand was reacted withNHS-biotin ester to provide the 5' biotinylated primers.

(iii) The primers were used for standard amplification, after which theamplimers were captured on avidinylated 96-well filter plates and washedto remove unreacted materials and contaminants.

(iv) The captured amplimers were hybridized with fluorescent analoglabeled oligonucleotide probes as described above and the amount oftarget sequence in the amplimers quantified.

Included in the present invention are such attachments of fluorescentoligonucleotides to other fluorescent or non-fluorescentoligonucleotides to immobilizing beads, filters, or activated plasticplates and done through enzymatic attachment such as ligation, orchemical attachment through such linkers as are described herein.

EXAMPLE 11 Uses of Fluorescence Resonance Energy. Transfer (FRET) toBroaden or Enhance the Uses of Fluorescent Nucleoside Analogs and Probes

Oligonucleotides can be synthesized or derivatized as described hereinwhich have two or more spectrally distinct, detectable labels, either byusing two or more nucleoside analogs with discrete fluorescence emissioncharacteristics, or by use of a covalently attached FRET acceptor, suchas is described hereinabove. FRET acceptors can also be used to enhanceor broaden the sensitivity of the detection for the fluorescent probes,if they are simply available in solution to act as acceptors of theprobe emission. For example, the excitation spectra of such dyes as thecoumarins, e.g., 7-amino-4-methylcoumarin-3-acetate,7-methyl-umbelliferone, the naphthalene and anthracene dyes, etc.,overlap the emission spectrum of oligomers constructed from thefluorescent nucleoside analogs, e.g., poly (FU), but not the oligomers'excitation spectrum. Such dyes as 7-amino-4-methylcoumarin-3-acetate maythus be used either (i) as a covalently attached FRET acceptor, e.g., byreacting the N-hydroxysuccinimide ester with prescribed amino groups onthe oligomer, or (ii) by simply adding the dye to a solution of theprobe to act as a FRET indicator of probe fluorescence. In addition tothe obvious advantages of providing a second fluorescent label to thehybridization probe, this methodology allows amplification of the probesignal through more efficient capture of the emitted light, reduction ofbackground light due to light scattering from excitation sources, anddetection at longer visible wavelengths.

EXAMPLE 12 RNase Amplification Method

First an RNA fluorescent probe is contacted with a DNA sample. The RNAfluorescent probe hybridizes to a target DNA sequence. RNase H onlydigests RNA:DNA hybrids, not ssRNA probes. The resulting fluorescentmonomers are released into solution, and a second RNA probe canhybridize to be digested. At the end of the experiment, the monomers areseparated from probes on standard membranes, and the amount of monomerreleased is measured by simple fluorometry. The specimens with no DNAfor hybrids will show no fluorescence.

It should be understood that the examples and embodiments describedherein are for illustrative purposes only and that various modificationsor changes in light thereof will be suggested m persons skilled in theart and are to be included within the spirit and purview of thisapplication and the scope of the appended claims.

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 3                                                  (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 39 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Chlamydia trachomatis                                           (C) INDIVIDUAL ISOLATE: L2/434/Bu                                             (G) CELL TYPE: Bacterium                                                      (vii) IMMEDIATE SOURCE:                                                       (A) LIBRARY: lambda 1059 recombinant                                          (B) CLONE: lamdba gt11/L2/33                                                  (viii) POSITION IN GENOME:                                                    (A) CHROMOSOME/SEGMENT: omp1l2 ORF                                            (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       AACGTTCGAGACGGACACCCCTTAGGACGACTTGGTTCG39                                     (2) INFORMATION FOR SEQ ID NO:2:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 39 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: transcribed DNA or RNA                                    (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: YES                                                          (ix) FEATURE:                                                                 (A) NAME/KEY: Complementary probe                                             (C) IDENTIFICATION METHOD: Hybridization to SEQ ID NO. 1                      (D) OTHER INFORMATION: Control for SEQ ID NO. 3                               (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                       TTGCAAGCTCTGCCTGTGGGGAATCCTGCTGAACCAAGC39                                     (2) INFORMATION FOR SEQ ID NO:3:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 39 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: transcribed DNA or RNA                                    (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: YES                                                          (ix) FEATURE:                                                                 (A) NAME/KEY: Analogous complementary probe                                   (C) IDENTIFICATION METHOD: Hybridization to SEQ ID NO. 1                      (D) OTHER INFORMATION: Analog to SEQ ID NO. 2                                 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                       TTGCNNGCTCTGCCTGTGGGGNNTCCTGCTGNNCCNNGC39                                     __________________________________________________________________________

I claim:
 1. A 5' universal end label for a nucleic acid sequence,wherein said label comprises a 5' homopolymer, said homopolymercomprising a fluorescent nucleoside analog not capable of forminghydrogen-bonded Watson-Crick base pairs, and a sequence complementary toa promoter region on a nucleic acid sequence, wherein said fluorescentnucleoside analog has the structure: ##STR3## wherein X₁ ═X₆ ═C orSi;X₂, X₃, X₄, X₅ ═C, S, N or Si; provided that at least one of X₁, X₂,X₃, X₄, X₅, or X₆ ═N; X₇ ═--CH--; R₄ is a reactive group derivatizablewith a detectable label wherein said reactive group is selected from thegroup consisting of NH₂, SH, ═O, and optionally, a linking moietyselected from the group consisting of an amide, a thioether, adisulfide, a combination of an amide a thioether or a disulfide, R₁--(CH₂)_(x) --R₂ and R₁ --R₂ (CH₂)_(x) --R₃, wherein x is an integerfrom 1 to 25 inclusive, and R₁, R₂, and R₃ are H, OH, alkyl, acyl,amide, thioether, or disulfide, and wherein said detectable label isselected from the group consisting of radioisotopes, fluorescent orchemiluminescent reporter molecules, antibodies, haptens, biotin,photobiotin, digoxigenin, fluorescent aliphatic amino groups, avidin,enzymes, and acridinium; R₅ is absent, H, or is part of an ethenolinkage with R₄ ; R₆ is H, NH₂, SH, or ═O; R₈ and R₉ are independentlyabsent, hydrogen, methyl, bromine, fluorine, or iodine; an alkyl oraromatic substituent, or an optional linking moiety selected from thegroup consisting of an amide, a thioether, a disulfide linkage, and acombination thereof; R₁₀ is hydrogen, an acid-sensitive/base-stableblocking group, or a phosphorus derivative; R₁₁ ═R₁₃ ═H; R₁₂ ishydrogen, OH, 3' amino, 3'-azido, 3'-thiol, 3'-unsaturated or a3'-phosphorus derivative; and R₁₄ is H, OH, or OR₃ where R₃ is areactive group, protecting group, or additional fluorophore; providedthat, excluded from such compound is any purine-like compound in which:(i) X1═X4═C; X2═X3═N; R4═NH2;R5═R8 which is absent; R6═H; R9is H orabsent; R10═H; and R12═R14═OH; or (ii) X1═C; X2═X3═X4═N; R4═NH2or H;R5═R8 which is absent; R6═NH2; R9 is H or is absent; R10═H; andR12═R14═OH; or (iii) R4 and R5 in combination form an etheno linkage;R6═R8═H; R9 is absent; X1═X3═C; and X2═X4═N; or (iv) X1═X2═C; X3═X4═N;R4═halogen or --S(CH₂)_(n) R, with n being an integer between 1-6 and Ris lower alkoxy, alkylthio, phenoxy, phenylthio, unsubstituted orsubstituted phenyl, --C═C--R', wherein R' is unsubstituted or on-, di-or trisubstituted phenyl; R9═R10═H; and R12═R14═acyloxy; or (v) R4═NH2or OH; R5 is absent; R9 is --COOH, --CONH2, --C(S)NH2, --C(NH)NH2, or--C(N-- NH2)NH2; X1═X2═X3═C; and X4═N.
 2. The universal end label,according to claim 1 further having a nucleic acid sequence disposedbetween and connected to said homopolymer sequence and said sequencecomplementary to a promoter region.
 3. The universal end label,according to claim 1, wherein said universal end label further comprisesa nucleic acid sequence complementary to a target sequence which isdifferent from said promoter region.
 4. A method for synthesizing afluorescent 5' end label probe comprising a 5' homopolymer, saidhomopolymer comprising a fluorescent nucleoside analog not capable offorming hydrogen-bonded Watson-Crick base pairs, according to claim 1and a sequence complementary to a promoter region or a nucleic acidsequence, said method comprising the steps of(a) restricting, with aspecific restriction enzyme, a sequence having a known promoter site andknown restriction site downstream from the known promoter site; (b)inserting a unique target sequence at the restriction site; (c)hybridizing a fluorescent nucleoside analog probe comprising a sequencecomplementary to the promoter of the inserted target sequence; and (d)extending the probe sequence from the hybridized promoter region, usinga nucleic acid polymerase, to synthesize a specific probe complementaryto the inserted target sequence.
 5. A method for increased sensitivityof detection of a target nucleotide sequence, said method comprising(a)chemically synthesizing a 5' universal end label according to claim 1;(b) enzymatically synthesizing a plurality of nucleic acid sequencescomplementary to a discreet segment of said target sequence, whereineach of said plurality of nucleic acid sequences comprises an end labelof step (a); (c) providing as a cocktail a mixture of the plurality of5' end label nucleic acid sequences; and (d) hybridizing the labelednucleic acid sequences to a target nucleic acid sequence.
 6. A methodfor determining the sequence of bases in a polynucleotide, said methodcomprising(a) synthesizing a plurality of nucleic acid fragments eachhaving different lengths, wherein each of the nucleic acid fragments hasattached thereto a 5' universal end label according to claim 1; (b)separating on a gel each of the different lengths of said nucleic acidfragments; and (c) detecting directly on the gel, each of said fragmentlengths.